Antibodies that bind to axl proteins

ABSTRACT

Antibodies that bind to AXL protein and variants thereof are described herein. AXL exhibits a distinct and limited expression pattern in normal adult tissue(s), and is aberrantly expressed in the cancers listed in Table I. Consequently, the MAbs of the invention provide a diagnostic composition for the treatment and management of cancer.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationNo. 62/163,264, filed 18 May 2015, the contents of which is incorporatedby reference in its entirety.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitled511582009840SEQLIST.TXT, created May 18, 2016 which is 32.4 kilobytes insize. The information in the electronic format of the Sequence Listingis incorporated by reference in its entirety.

STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH

Not applicable.

FIELD OF THE INVENTION

The invention described herein relates to antibodies and antigen-bindingfragments thereof, that bind proteins, termed AXL. The invention furtherrelates to prognostic, prophylactic, diagnostic and companiontherapeutic methods and compositions useful in the treatment of cancersthat express AXL.

BACKGROUND OF THE INVENTION

It is estimated that 1,660,290 men and women (854,790 men and 805,500women) will be diagnosed with and 580,350 men and women will die ofcancer of all sites in 2013. From 2006-2010, the median age at diagnosisfor cancer of all sites was 66 years of age. The age-adjusted incidencerate was 463.0 per 100,000 men and women per year. These rates are basedon cases diagnosed in 2006-2010 from 18 SEER geographic areas. From2006-2010, the median age at death for cancer of all sites was 72 yearsof age. The age-adjusted death rate was 176.4 per 100,000 men and womenper year. These rates are based on patients who died in 2006-2010 in theUS. The overall 5-year relative survival for 2003-2009 from 18 SEERgeographic areas was 65.8%.

Non-Hodgkin lymphomas (NHLs) can occur at any age and are often markedby lymph nodes that are larger than normal, fever, and weight loss.There are many different types of non-Hodgkin lymphoma. These types canbe divided into aggressive (fast-growing) and indolent (slow-growing)types, and they can be formed from either B-cells or T-cells. B-cellnon-Hodgkin lymphomas include Burkitt lymphoma, chronic lymphocyticleukemia/small lymphocytic lymphoma (CLL/SLL), diffuse large B-celllymphoma, follicular lymphoma, immunoblastic large cell lymphoma,precursor B-lymphoblastic lymphoma, and mantle cell lymphoma. T-cellnon-Hodgkin lymphomas include mycosis fungoides, anaplastic large celllymphoma, and precursor T-lymphoblastic lymphoma. Lymphomas that occurafter bone marrow or stem cell transplantation are usually B-cellnon-Hodgkin lymphomas. Prognosis and treatment depend on the stage andtype of disease.

It is estimated that 69,740 men and women (37,600 men and 32,140 women)will be diagnosed with and 19,020 men and women will die of non-Hodgkinlymphoma in 2013. From 2006-2010, the median age at diagnosis fornon-Hodgkin lymphoma was 66 years of age. The age-adjusted incidencerate was 19.7 per 100,000 men and women per year. These rates are basedon cases diagnosed in 2006-2010 from 18 SEER geographic areas. From2006-2010, the median age at death for non-Hodgkin lymphoma was 76 yearsof age. The age-adjusted death rate was 6.4 per 100,000 men and womenper year. These rates are based on patients who died in 2006-2010 in theUS. The overall 5-year relative survival for 2003-2009 from 18 SEERgeographic areas was 69.0%.

Leukemias are cancers that start in blood-forming tissue such as thebone marrow and causes large numbers of blood cells to be produced andenter the bloodstream. The major leukemias are comprised of AcuteLymphoblastic (ALL), Acute Myeloid (AML), Chronic Lymphocytic (CLL),Chronic Myelogenous (CML), and Hairy Cell (CLL) Leukemia.

For these leukemias as a group, it is estimated that 48,610 men andwomen (27,880 men and 20,730 women) will be diagnosed with and 23,720men and women will die of leukemia in 2013. From 2006-2010, the medianage at diagnosis for leukemia was 66 years of age. The age-adjustedincidence rate was 12.8 per 100,000 men and women per year. These ratesare based on cases diagnosed in 2006-2010 from 18 SEER geographic areas.From 2006-2010, the median age at death for leukemia was 75 years ofage. The age-adjusted death rate was 7.1 per 100,000 men and women peryear. These rates are based on patients who died in 2006-2010 in the US.The overall 5-year relative survival for 2003-2009 from 18 SEERgeographic areas was 56.0%.

CLL is the second most common type of leukemia in adults and it usuallygets worse slowly. It often occurs during or after middle age and itrarely occurs in children. Patients with early-stage CLL are not treatedwith chemotherapy until they become symptomatic or display evidence ofrapid progression of disease. Early initiation of chemotherapy hasfailed to show benefit in CLL and may even increase mortality. Whenchemotherapy is initiated, the nucleoside analogue fludarabine is themost commonly used first-line therapy in CLL. Combination regimens haveshown improved response rates in several clinical trials and include thefollowing: Fludarabine, cyclophosphamide, and rituximab (FCR);Pentostatin, cyclophosphamide, and rituximab (PCR); Fludarabine,cyclophosphamide, and mitoxantrone (FCM); Cyclophosphamide, vincristine,and prednisone (CVP); Cyclophosphamide, doxorubicin, vincristine, andprednisone (CHOP). It is estimated that 15,680 men and women (9,720 menand 5,960 women) will be diagnosed with and 4,580 men and women will dieof chronic lymphocytic leukemia in 2013. From 2006-2010, the median ageat diagnosis for chronic lymphocytic leukemia was 71 years of age. Theage-adjusted incidence rate was 4.3 per 100,000 men and women per year.These rates are based on cases diagnosed in 2006-2010 from 18 SEERgeographic areas. From 2006-2010, the median age at death for chroniclymphocytic leukemia was 79 years of age. The age-adjusted death ratewas 1.4 per 100,000 men and women per year. These rates are based onpatients who died in 2006-2010 in the US. The overall 5-year relativesurvival for 2003-2009 from 18 SEER geographic areas was 79.2%.

Acute myeloid leukemia (AML) is the most common type of acute leukemiaamong adults. Current treatment of AML should be sufficiently aggressiveto achieve complete remission (CR) because partial remission offers nosubstantial survival benefit. Remission rates in adult AML are inverselyrelated to age, with an expected remission rate of more than 65% forthose younger than 60 years. Data suggest that once attained, durationof remission may be shorter in older patients. Patients that express theprogenitor cell antigen CD34 and/or the P-glycoprotein (MDR1 geneproduct) have an inferior outcome. Cytogenetic analysis provides some ofthe strongest prognostic information available, predicting outcome ofboth remission induction and post remission therapy. Cytogeneticabnormalities that indicate a good prognosis include t(8; 21), inv(16)or t(16;16), and t(15;17). Normal cytogenetics portends average-riskAML. Patients with AML that is characterized by deletions of the longarms or monosomies of chromosomes 5 or 7; by translocations orinversions of chromosome 3, t(6; 9), t(9; 22); or by abnormalities ofchromosome 11q23 have particularly poor prognoses with chemotherapy. Itis estimated that 14,590 men and women (7,820 men and 6,770 women) willbe diagnosed with and 10,370 men and women will die of acute myeloidleukemia in 2013. From 2006-2010, the median age at diagnosis for acutemyeloid leukemia was 67 years of age. The age-adjusted incidence ratewas 3.7 per 100,000 men and women per year. These rates are based oncases diagnosed in 2006-2010 from 18 SEER geographic areas. From2006-2010, the median age at death for acute myeloid leukemia was 72years of age. The age-adjusted death rate was 2.8 per 100,000 men andwomen per year. These rates are based on patients who died in 2006-2010in the US. The overall 5-year relative survival for 2003-2009 from 18SEER geographic areas was 24.2%. Note, all general cancer informationwas obtained from the NCI website (www.cancer.gov) and all statisticsare based on SEER incidence and NCHS mortality statistics found within:Howlader N., et. al., SEER Cancer Statistics Review, 1975-2010, NationalCancer Institute. Bethesda, Md., http://seer.cancer.gov/csr/1975_2010/,based on November 2012 SEER data submission, posted to the SEER website, 2013.

The therapeutic utility of monoclonal antibodies (mAbs) (G. Kohler andC. Milstein, Nature 256:495-497 (1975)) is being realized. Monoclonalantibodies have now been approved as therapies in transplantation,cancer, infectious disease, cardiovascular disease and inflammation.Different isotypes have different effector functions. Such differencesin function are reflected in distinct 3-dimensional structures for thevarious immunoglobulin isotypes (P. M. Alzari et al., Annual Rev.Immunol., 6:555-580 (1988)).

Furthermore, recent years, monoclonal antibodies are reported to be usedfor Companion diagnostics (Sai-Hong Ignatius Ou, et al, Front Oncol.2014; 4: 58). Zhang firstly reports that activation of the AXL kinasecauses resistance to EGFR-targeted therapy in lung cancer (NatureGenetics 44,852-860(2012)) and AF154AXL rabbit polyAb (R&D Systems) isused for detecting AXL in lung cancer patients (US 20140121126).

Because mice are convenient for immunization and recognize most humanantigens as foreign, mAbs against human targets with therapeuticpotential have typically been of murine origin. However, murine mAbshave inherent disadvantages as human therapeutics. They require morefrequent dosing as mAbs have a shorter circulating half-life in humansthan human antibodies. More critically, the repeated administration ofmurine antibodies to the human immune system causes the human immunesystem to respond by recognizing the mouse protein as a foreign andgenerating a human anti-mouse antibody (HAMA) response. Such a HAMAresponse may result in allergic reaction and the rapid clearing of themurine antibody from the system thereby rendering the treatment bymurine antibody useless. To avoid such affects, attempts to create humanimmune systems within mice have been attempted.

Initial attempts hoped to create transgenic mice capable of respondingto antigens with antibodies having human sequences (See Bruggemann etal., Proc. Nat'l. Acad. Sci. USA 86:6709-6713 (1989)), but were limitedby the amount of DNA that could be stably maintained by availablecloning vehicles. The use of yeast artificial chromosome (YAC) cloningvectors led the way to introducing large germline fragments of human Iglocus into transgenic mammals. Essentially a majority of the human V, D,and J region genes arranged with the same spacing found in the humangenome and the human constant regions were introduced into mice usingYACs. One such transgenic mouse strain is known as XenoMouse® mice andis commercially available from Amgen Fremont, Inc. (Fremont Calif.).

Additionally, antibodies can be prepared using VelocImmune transgenicmice into which genomic sequences bearing endogenous mouse variablesegments at the immunoglobulin heavy chain (VH, DH, and JH segments)and/or kappa light chain (VK and JK) loci have been replaced, in wholeor in part, with human genomic sequences bearing unrearranged germlinevariable segments of the human immunoglobulin heavy chain (VH, DH, andJH) and/or kappa light chain (VK and JK) loci (Regeneron, Tarrytown,N.Y.). See, for example, U.S. Pat. Nos. 6,586,251, 6,596,541, 7,105,348,6,528,313, 6,638,768, and 6,528,314.

SUMMARY OF THE INVENTION

The invention provides antibodies and antigen-binding fragments thereofthat bind to AXL proteins and polypeptide fragments of AXL proteins. Insome embodiments, the invention comprises fully human antibodies.

The invention further provides various immunogenic or therapeuticcompositions, such as antibodies labelled with an imaging agent, andstrategies for diagnosing, and treating cancers that express AXL such ascancers of tissues listed in Table I.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. The cDNA (SEQ ID NO: 1) and amino acid sequence (SEQ ID NO: 2)of AXL is shown in FIG. 1. The start methionine is underlined. The openreading frame extends from nucleic acid 191-2875 including the stopcodon.

FIG. 2A. Nucleic Acid and Amino Acid sequences of AXL antibodies. ThecDNA (SEQ ID NO: 3) and amino acid sequence (SEQ ID NO: 4) of V77-2a37.1heavy chain. Double-underlined is the heavy chain variable region, andunderlined is the heavy chain mouse IgG1 constant region.

FIG. 2B. Nucleic Acid and Amino Acid sequences of AXL antibodies. ThecDNA (SEQ ID NO: 5) and amino acid sequence (SEQ ID NO: 6) of V77-2a37.1light chain. Double-underlined is the light chain variable region, andunderlined is the mouse kappa constant region.

FIG. 3A. Amino Acid sequences of V77-2a37.1 antibodies. The amino acidsequence of the V77-2a37.1 heavy chain (SEQ ID NO: 7). The CDR regions(based on kabat numbering) are Italicized. Double-underlined is theheavy chain variable region (SEQ ID NO: 8), and underlined is the mouseIgG1 constant region.

FIG. 3B. Amino Acid sequences of V77-2a37.1 antibodies. The amino acidsequence of the V77-2a37.1 light chain (SEQ ID NO: 9). The CDR regions(based on kabat numbering) are Italicized. Double-underlined is thelight chain variable region (SEQ ID NO: 10), and underlined is the mousekappa constant region.

FIG. 4A. Alignment of V77-2a37.1 heavy chain variable region to human Iggermline.

FIG. 4B. Alignment of V77-2a37.1 light chain variable region to human Iggermline.

FIG. 5A-FIG. 5J. Detection of AXL protein in cancer patient specimens byIHC. FIG. 5A and FIG. 5B show sarcoma cancer specimens. FIG. 5C and FIG.5D show pancreatic cancer specimens. FIG. 5E and FIG. 5F show melanomacancer specimens. FIG. 5G and FIG. 5H show ovarian cancer specimens.FIG. 5I and FIG. 5J show lung cancer specimens. AXL MAb denotedV77-2a37.1 is shown in FIGS. 5A, C, E, G, and I. A Negative controlantibody is shown in FIGS. 5B, D, F, H, and J.

FIG. 6A(i), FIG. 6A(ii), and FIG. 6A(iii). Detection of AXL expressionin FFPE cell block samples from three cell lines, FIG. 6A(i) NCI-H292cells, FIG. 6A(ii) HCC827 cells, and FIG. 6A(iii) NCI-H727 cells, by RNAISH.

FIG. 6B(i), FIG. 6B(ii), FIG. 6B(iii), FIG. 6B(iv), FIG. 6B(v), and FIG.6B(vi). Detection of AXL expression in FFPE cell block samples fromthree cell lines (NCI-H292 cells, HCC827 cells, and NCI-H727 cells) byIHC using a Cell Signaling rabbit monoclonal antibody [C89E7] (FIGS.6B(i), 6B(ii), and 6B(iii)) and a R&D goat polyclonal antibody [AF154](FIGS. 6B(iv), 6B(v), and 6B(vi)). FIG. 6B(i): NCI-H292 cells withanti-AXL antibody [C89E7]. FIG. 6B(ii): HCC827 cells with anti-AXLantibody [C89E7]. FIG. 6B(iii): NCI-H727 cells with anti-AXL antibody[C89E7]. FIG. 6B(iv): NCI-H292 cells with anti-AXL antibody [AF154].FIG. 6B(v): HCC827 cells with anti-AXL antibody [AF154]. FIG. 6B(vi):NCI-H727 with anti-AXL antibody [AF154].

FIGS. 7A-7L. Anti-AXL antibody immunohistochemistry (IHC) comparison inAXL-negative cancer specimens. Detection of AXL non-specific IHCstaining in breast (FIGS. 7A-7D), hepatocellular (FIGS. 7E-7H), andcolon (FIGS. 7I-7L) carcinoma specimens. IHC assay performed withV77-2a37.1 (FIGS. 7A, 7E, 7I), M77-297b81.1.1 (FIGS. 7B, 7F, 7J), AXL(C89E7) (FIGS. 7C, 7G, 7K), and mouse IgG1 negative control antibody(FIGS. 7D, 7H, 7L). The results showed more non-specific IHC stainingwith AXL (C89E7) in cancer specimens with limited AXL mRNA expression(FIGS. 7C, 7G, 7K; see arrows).

DETAILED DESCRIPTION OF THE INVENTION Outline of Sections

-   I.) Definitions-   II.) AXL Antibodies-   III.) Diagnosis of Cancer(s) Expressing AXL-   IV.) Treatment of Cancer(s) Expressing AXL-   V.) AXL as a Target for Antibody-based Therapy-   VI.) AXL ADC Cocktails-   VII.) Combination Therapy-   VIII.) Kits/Articles of Manufacture

I.) Definitions

Unless otherwise defined, all terms of art, notations and otherscientific terms or terminology used herein are intended to have themeanings commonly understood by those of skill in the art to which thisinvention pertains. In some cases, terms with commonly understoodmeanings are defined herein for clarity and/or for ready reference, andthe inclusion of such definitions herein should not necessarily beconstrued to represent a substantial difference over what is generallyunderstood in the art. Many of the techniques and procedures describedor referenced herein are well understood and commonly employed usingconventional methodology by those skilled in the art, such as, forexample, the widely utilized molecular cloning methodologies describedin Sambrook et al., Molecular Cloning: A Laboratory Manual 2nd. edition(1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. Asappropriate, procedures involving the use of commercially available kitsand reagents are generally carried out in accordance with manufacturerdefined protocols and/or parameters unless otherwise noted.

When a trade name is used herein, reference to the trade name alsorefers to the product formulation, the generic drug, and the activepharmaceutical ingredient(s) of the trade name product, unless otherwiseindicated by context.

The terms “advanced cancer”, “locally advanced cancer”, “advanceddisease” and “locally advanced disease” mean cancers that have extendedthrough the relevant tissue capsule, and are meant to include stage Cdisease under the American Urological Association (AUA) system, stageC1-C2 disease under the Whitmore-Jewett system, and stage T3-T4 and N+disease under the TNM (tumor, node, metastasis) system. In general,surgery is not recommended for patients with locally advanced disease,and these patients have substantially less favorable outcomes comparedto patients having clinically localized (organ-confined) cancer.

“Altering the native glycosylation pattern” is intended for purposesherein to mean deleting one or more carbohydrate moieties found innative sequence AXL (either by removing the underlying glycosylationsite or by deleting the glycosylation by chemical and/or enzymaticmeans), and/or adding one or more glycosylation sites that are notpresent in the native sequence AXL. In addition, the phrase includesqualitative changes in the glycosylation of the native proteins,involving a change in the nature and proportions of the variouscarbohydrate moieties present.

The term “analog” refers to a molecule which is structurally similar orshares similar or corresponding attributes with another molecule (e.g. aAXL-related protein). For example, an analog of a AXL protein can bespecifically bound by an antibody or T cell that specifically binds toAXL.

The term “antibody” is used in the broadest sense unless clearlyindicated otherwise. Therefore, an “antibody” can be naturally occurringor man-made such as monoclonal antibodies produced by conventionalhybridoma technology. AXL antibodies comprise monoclonal and polyclonalantibodies as well as fragments containing the antigen-binding domainand/or one or more complementarity determining regions of theseantibodies. As used herein, the term “antibody” refers to any form ofantibody or fragment thereof that specifically binds AXL and/or exhibitsthe desired biological activity and specifically covers monoclonalantibodies (including full length monoclonal antibodies), polyclonalantibodies, multispecific antibodies (e.g., bispecific antibodies), andantibody fragments so long as they specifically bind AXL and/or exhibitthe desired biological activity. Any specific antibody can be used inthe methods and compositions provided herein. Thus, in one embodimentthe term “antibody” encompasses a molecule comprising at least onevariable region from a light chain immunoglobulin molecule and at leastone variable region from a heavy chain molecule that in combination forma specific binding site for the target antigen. In one embodiment, theantibody is an IgG antibody. For example, the antibody is a IgG1, IgG2,IgG3, or IgG4 antibody. The antibodies useful in the present methods andcompositions can be generated in cell culture, in phage, or in variousanimals, including but not limited to cows, rabbits, goats, mice, rats,hamsters, guinea pigs, sheep, dogs, cats, monkeys, chimpanzees, andapes. Therefore, in one embodiment, an antibody of the present inventionis a mammalian antibody. Phage techniques can be used to isolate aninitial antibody or to generate variants with altered specificity oravidity characteristics. Such techniques are routine and well known inthe art. In one embodiment, the antibody is produced by recombinantmeans known in the art. For example, a recombinant antibody can beproduced by transfecting a host cell with a vector comprising a DNAsequence encoding the antibody. One or more vectors can be used totransfect the DNA sequence expressing at least one VL and one VH regionin the host cell. Exemplary descriptions of recombinant means ofantibody generation and production include Delves, ANTIBODY PRODUCTION:ESSENTIAL TECHNIQUES (Wiley, 1997); Shephard, et al., MONOCLONALANTIBODIES (Oxford University Press, 2000); Goding, MONOCLONALANTIBODIES: PRINCIPLES AND PRACTICE (Academic Press, 1993); and CURRENTPROTOCOLS IN IMMUNOLOGY (John Wiley & Sons, most recent edition). Anantibody of the present invention can be modified by recombinant meansto increase efficacy of the antibody in mediating the desired function.Thus, it is within the scope of the invention that antibodies can bemodified by substitutions using recombinant means. Typically, thesubstitutions will be conservative substitutions. For example, at leastone amino acid in the constant region of the antibody can be replacedwith a different residue. See, e.g., U.S. Pat. No. 5,624,821, U.S. Pat.No. 6,194,551, Application No. WO 9958572; and Angal, et al., Mol.Immunol. 30: 105-08 (1993). The modification in amino acids includesdeletions, additions, and substitutions of amino acids. In some cases,such changes are made to reduce undesired activities, e.g.,complement-dependent cytotoxicity. Frequently, the antibodies arelabeled by joining, either covalently or non-covalently, a substancewhich provides for a detectable signal. A wide variety of labels andconjugation techniques are known and are reported extensively in boththe scientific and patent literature. These antibodies can be screenedfor binding to normal or defective AXL. See e.g., ANTIBODY ENGINEERING:A PRACTICAL APPROACH (Oxford University Press, 1996). Suitableantibodies with the desired biologic activities can be identified usingthe following in vitro assays including but not limited to:proliferation, migration, adhesion, soft agar growth, angiogenesis,cell-cell communication, apoptosis, transport, signal transduction, andthe following in vivo assays such as the inhibition of tumor growth. Theantibodies provided herein can also be useful in diagnosticapplications. As capture or non-neutralizing antibodies, they can bescreened for the ability to bind to the specific antigen withoutinhibiting the receptor-binding or biological activity of the antigen.As neutralizing antibodies, the antibodies can be useful in competitivebinding assays. They can also be used to quantify the AXL or itsreceptor.

The term “antigen-binding portion” or “antibody fragment” or “antigenbinding fragment” of an antibody (or simply “antibody portion”), as usedherein, refers to one or more fragments of a AXL antibody that retainthe ability to specifically bind to an antigen (e.g., AXL and variants;FIG. 1). It has been shown that the antigen-binding function of anantibody can be performed by fragments of a full-length antibody.Examples of binding fragments encompassed within the term“antigen-binding portion” of an antibody include (i) a Fab fragment, amonovalent fragment consisting of the V_(L), V_(H), C_(L) and C_(H1)domains; (ii) a F(ab′)₂ fragment, a bivalent fragment comprising two Fabfragments linked by a disulfide bridge at the hinge region; (iii) a Fdfragment consisting of the V_(H) and C_(H1) domains; (iv) a Fv fragmentconsisting of the V_(L) and V_(H) domains of a single arm of anantibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546),which consists of a V_(H) domain; and (vi) an isolated complementarilydetermining region (CDR). Furthermore, although the two domains of theFv fragment, V_(L) and V_(H), are coded for by separate genes, they canbe joined, using recombinant methods, by a synthetic linker that enablesthem to be made as a single protein chain in which the V_(L) and V_(H)regions pair to form monovalent molecules (known as single chain Fv(scFv); see e.g., Bird et al. (1988) Science 242:423-426; and Huston etal. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single chainantibodies are also intended to be encompassed within the term“antigen-binding portion” of an antibody. These antibody fragments areobtained using conventional techniques known to those with skill in theart, and the fragments are screened for utility in the same manner asare intact antibodies.

As used herein, any form of the “antigen” can be used to generate anantibody that is specific for AXL. Thus, the eliciting antigen may be asingle epitope, multiple epitopes, or the entire protein alone or incombination with one or more immunogenicity enhancing agents known inthe art. The eliciting antigen may be an isolated full-length protein, acell surface protein (e.g., immunizing with cells transfected with atleast a portion of the antigen), or a soluble protein (e.g., immunizingwith only the extracellular domain portion of the protein). The antigenmay be produced in a genetically modified cell. The DNA encoding theantigen may be genomic or non-genomic (e.g., cDNA) and encodes at leasta portion of the extracellular domain. As used herein, the term“portion”, in the context of an antigen, refers to the minimal number ofamino acids or nucleic acids, as appropriate, to constitute animmunogenic epitope of the antigen of interest. Any genetic vectorssuitable for transformation of the cells of interest may be employed,including but not limited to adenoviral vectors, plasmids, and non-viralvectors, such as cationic lipids. In one embodiment, the antibody of themethods and compositions herein specifically bind at least a portion ofthe extracellular domain of the AXL of interest.

The antibodies or antigen binding fragments thereof provided herein maybe conjugated to a “bioactive agent.” As used herein, the term“bioactive agent” refers to any synthetic or naturally occurringcompound that binds the antigen and/or enhances or mediates a desiredbiological effect to enhance cell-killing toxins. In one embodiment, thebinding fragments useful in the present invention are biologicallyactive fragments. As used herein, the term “biologically active” refersto an antibody or antibody fragment that is capable of binding thedesired antigenic epitope and directly or indirectly exerting a biologiceffect. Direct effects include, but are not limited to the modulation,stimulation, and/or inhibition of a growth signal, the modulation,stimulation, and/or inhibition of an anti-apoptotic signal, themodulation, stimulation, and/or inhibition of an apoptotic or necroticsignal, modulation, stimulation, and/or inhibition the ADCC cascade, andmodulation, stimulation, and/or inhibition the CDC cascade.

“Bispecific” antibodies are also useful in the present methods andcompositions. As used herein, the term “bispecific antibody” refers toan antibody, typically a monoclonal antibody, having bindingspecificities for at least two different antigenic epitopes. In oneembodiment, the epitopes are from the same antigen. In anotherembodiment, the epitopes are from two different antigens. Methods formaking bispecific antibodies are known in the art. For example,bispecific antibodies can be produced recombinantly using theco-expression of two immunoglobulin heavy chain/light chain pairs. See,e.g., Milstein et al., Nature 305:537-39 (1983). Alternatively,bispecific antibodies can be prepared using chemical linkage. See, e.g.,Brennan, et al., Science 229:81 (1985). Bispecific antibodies includebispecific antibody fragments. See, e.g., Hollinger, et al., Proc. Natl.Acad. Sci. U.S.A. 90:6444-48 (1993), Gruber, et al., J. Immunol.152:5368 (1994).

The monoclonal antibodies described herein specifically include“chimeric” antibodies in which a portion of the heavy and/or light chainis identical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is identical withor homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they specifically bindthe target antigen and/or exhibit the desired biological activity (U.S.Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)).

The term “Chemotherapeutic Agent” refers to all chemical compounds thatare effective in inhibiting tumor growth. Non-limiting examples ofchemotherapeutic agents include alkylating agents; for example, nitrogenmustards, ethyleneimine compounds and alkyl sulphonates;antimetabolites, for example, folic acid, purine or pyrimidineantagonists; mitotic inhibitors, for example, anti-tubulin agents suchas vinca alkaloids, auristatins and derivatives of podophyllotoxin;cytotoxic antibiotics; compounds that damage or interfere with DNAexpression or replication, for example, DNA minor groove binders; andgrowth factor receptor antagonists. In addition, chemotherapeutic agentsinclude cytotoxic agents (as defined herein), antibodies, biologicalmolecules and small molecules.

The term “compound” refers to and encompasses the chemical compounditself as well as, whether explicitly stated or not, and unless thecontext makes clear that the following are to be excluded: amorphous andcrystalline forms of the compound, including polymorphic forms, wherethese forms may be part of a mixture or in isolation; free acid and freebase forms of the compound, which are typically the forms shown in thestructures provided herein; isomers of the compound, which refers tooptical isomers, and tautomeric isomers, where optical isomers includeenantiomers and diastereomers, chiral isomers and non-chiral isomers,and the optical isomers include isolated optical isomers as well asmixtures of optical isomers including racemic and non-racemic mixtures;where an isomer may be in isolated form or in a mixture with one or moreother isomers; isotopes of the compound, including deuterium- andtritium-containing compounds, and including compounds containingradioisotopes, including therapeutically- and diagnostically-effectiveradioisotopes; multimeric forms of the compound, including dimeric,trimeric, etc. forms; salts of the compound, preferably pharmaceuticallyacceptable salts, including acid addition salts and base addition salts,including salts having organic counterions and inorganic counterions,and including zwitterionic forms, where if a compound is associated withtwo or more counterions, the two or more counterions may be the same ordifferent; and solvates of the compound, including hemisolvates,monosolvates, disolvates, etc., including organic solvates and inorganicsolvates, said inorganic solvates including hydrates; where if acompound is associated with two or more solvent molecules, the two ormore solvent molecules may be the same or different. In some instances,reference made herein to a compound of the invention will include anexplicit reference to one or of the above forms, e.g., salts and/orsolvates; however, this reference is for emphasis only, and is not to beconstrued as excluding other of the above forms as identified above.

The terms “complementarity determining region,” and “CDR,” are known inthe art to refer to non-contiguous sequences of amino acids withinantibody variable regions, which confer antigen specificity and bindingaffinity. In general, there are three (3) CDRs in each heavy chainvariable region (CDR-H1, CDR-H2, CDR-H3) and three (3) CDRs in eachlight chain variable region (CDR-L1, CDR-L2, CDR-L3).

The precise amino acid sequence boundaries of a given CDR can be readilydetermined using any of a number of well-known schemes, including thosedescribed by Kabat et al. (1991), “Sequences of Proteins ofImmunological Interest,” 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md. (“Kabat” numbering scheme),Al-Lazikani et al., (1997) JMB 273,927-948 (“Chothia” numbering scheme),MacCallum et al., J. Mol. Biol. 262:732-745 (1996), “Antibody-antigeninteractions: Contact analysis and binding site topography,” J. Mol.Biol. 262, 732-745.” (Contact” numbering scheme), Lefranc M P et al.,“IMGT unique numbering for immunoglobulin and T cell receptor variabledomains and Ig superfamily V-like domains,” Dev Comp Immunol, 2003January; 27 (1):55-77 (“IMGT” numbering scheme), and Honegger A andPlückthun A, “Yet another numbering scheme for immunoglobulin variabledomains: an automatic modeling and analysis tool,” J Mol Biol, 2001 Jun.8; 309 (3):657-70, (AHo numbering scheme).

The boundaries of a given CDR may vary depending on the scheme used foridentification. For example, the Kabat scheme is based structuralalignments, while the Chothia scheme is based on structural information.Numbering for both the Kabat and Chothia schemes is based upon the mostcommon antibody region sequence lengths, with insertions accommodated byinsertion letters, for example, “30a,” and deletions appearing in someantibodies. The two schemes place certain insertions and deletions(“indels”) at different positions, resulting in differential numbering.The Contact scheme is based on analysis of complex crystal structuresand is similar in many respects to the Chothia numbering scheme. TableV, infra, lists the positions of CDR-L1, CDR-L2, CDR-L3 and CDR-H1,CDR-H2, CDR-H3 as identified by the Kabat, Chothia, and Contact schemes,respectively. For CDR-H1, residue numbering is given listed using boththe Kabat and Chothia numbering schemes.

Thus, unless otherwise specified, the terms “CDR” and “complementarydetermining region” of a given antibody or region thereof, such as avariable region, as well as individual CDRs (e.g., “CDR-H1, CDR-H2) ofthe antibody or region thereof, should be understood to encompass thecomplementary determining region as defined by any of the known schemesdescribed herein above. In some instances, the scheme for identificationof a particular CDR or CDRs is specified, such as the CDR as defined bythe Kabat, Chothia, or Contact method. In other cases, the particularamino acid sequence of a CDR is given.

As used herein, the term “conservative substitution” refers tosubstitutions of amino acids are known to those of skill in this art andmay be made generally without altering the biological activity of theresulting molecule. Those of skill in this art recognize that, ingeneral, single amino acid substitutions in non-essential regions of apolypeptide do not substantially alter biological activity (see, e.g.,Watson, et al., MOLECULAR BIOLOGY OF THE GENE, The Benjamin/CummingsPub. Co., p. 224 (4th Edition 1987)). Such exemplary substitutions arepreferably made in accordance with those set forth in Table II andTable(s) III(a-b). For example, such changes include substituting any ofisoleucine (I), valine (V), and leucine (L) for any other of thesehydrophobic amino acids; aspartic acid (D) for glutamic acid (E) andvice versa; glutamine (Q) for asparagine (N) and vice versa; and serine(S) for threonine (T) and vice versa. Other substitutions can also beconsidered conservative, depending on the environment of the particularamino acid and its role in the three-dimensional structure of theprotein. For example, glycine (G) and alanine (A) can frequently beinterchangeable, as can alanine (A) and valine (V). Methionine (M),which is relatively hydrophobic, can frequently be interchanged withleucine and isoleucine, and sometimes with valine. Lysine (K) andarginine (R) are frequently interchangeable in locations in which thesignificant feature of the amino acid residue is its charge and thediffering pK's of these two amino acid residues are not significant.Still other changes can be considered “conservative” in particularenvironments (see, e.g. Table III(a) herein; pages 13-15 “Biochemistry”2nd ED. Lubert Stryer ed (Stanford University); Henikoff et al., PNAS1992 Vol 89 10915-10919; Lei et al., J Biol Chem 1995 May 19; 270(20):11882-6). Other substitutions are also permissible and may bedetermined empirically or in accord with known conservativesubstitutions.

As used herein, the term “diabodies” refers to small antibody fragmentswith two antigen-binding sites, which fragments comprise a heavy chainvariable domain (V_(H)) connected to a light chain variable domain(V_(L)) in the same polypeptide chain (V_(H)-V_(L)). By using a linkerthat is too short to allow pairing between the two domains on the samechain, the domains are forced to pair with the complementary domains ofanother chain and create two antigen-binding sites. Diabodies aredescribed more fully in, e.g., EP 404,097; WO 93/11161; and Hollinger etal., Proc. Natl. Acad. Sci. USA 90:6444-48 (1993).

The term “deplete,” in the context of the effect of a AXL binding agenton AXL-expressing cells, refers to a reduction in the number of orelimination of the AXL-expressing cells.

The term “gene product” is used herein to indicate a peptide/protein ormRNA. For example, a “gene product of the invention” is sometimesreferred to herein as a “cancer amino acid sequence”, “cancer protein”,“protein of a cancer listed in Table I”, a “cancer mRNA”, “mRNA of acancer listed in Table I”, etc. In one embodiment, the cancer protein isencoded by a nucleic acid of FIG. 1. The cancer protein can be afragment, or alternatively, be the full-length protein encoded bynucleic acids of FIG. 1. In one embodiment, a cancer amino acid sequenceis used to determine sequence identity or similarity. In anotherembodiment, the sequences are naturally occurring allelic variants of aprotein encoded by a nucleic acid of FIG. 1. In another embodiment, thesequences are sequence variants as further described herein.

“Heteroconjugate” antibodies are useful in the present methods andcompositions. As used herein, the term “heteroconjugate antibody” refersto two covalently joined antibodies. Such antibodies can be preparedusing known methods in synthetic protein chemistry, including usingcrosslinking agents. See, e.g., U.S. Pat. No. 4,676,980.

The term “homolog” refers to a molecule which exhibits homology toanother molecule, by for example, having sequences of chemical residuesthat are the same or similar at corresponding positions.

In one embodiment, the antibody provided herein is a “human antibody.”As used herein, the term “human antibody” refers to an antibody in whichessentially the entire sequences of the light chain and heavy chainsequences, including the complementary determining regions (CDRs), arefrom human genes. In one embodiment, human monoclonal antibodies areprepared by the trioma technique, the human B-cell technique (see, e.g.,Kozbor, et al., Immunol. Today 4: 72 (1983), EBV transformationtechnique (see, e.g., Cole et al. MONOCLONAL ANTIBODIES AND CANCERTHERAPY 77-96 (1985)), or using phage display (see, e.g., Marks et al.,J. Mol. Biol. 222:581 (1991)). In a specific embodiment, the humanantibody is generated in a transgenic mouse. Techniques for making suchpartially to fully human antibodies are known in the art and any suchtechniques can be used. According to one particularly preferredembodiment, fully human antibody sequences are made in a transgenicmouse engineered to express human heavy and light chain antibody genes.An exemplary description of preparing transgenic mice that produce humanantibodies found in Application No. WO 02/43478 and U.S. Pat. No.6,657,103 (Abgenix) and its progeny. B cells from transgenic mice thatproduce the desired antibody can then be fused to make hybridoma celllines for continuous production of the antibody. See, e.g., U.S. Pat.Nos. 5,569,825; 5,625,126; 5,633,425; 5,661,016; and 5,545,806; andJakobovits, Adv. Drug Del. Rev. 31:33-42 (1998); Green, et al., J. Exp.Med. 188:483-95 (1998).

As used herein, the term “humanized antibody” refers to forms ofantibodies that contain sequences from non-human (e.g., murine)antibodies as well as human antibodies. Such antibodies are chimericantibodies which contain minimal sequence derived from non-humanimmunoglobulin. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin and all or substantially all ofthe FR regions are those of a human immunoglobulin sequence. Thehumanized antibody optionally also will comprise at least a portion ofan immunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. See e.g., Cabilly U.S. Pat. No. 4,816,567; Queen et al.(1989) Proc. Nat'l Acad. Sci. USA 86:10029-10033; and ANTIBODYENGINEERING: A PRACTICAL APPROACH (Oxford University Press 1996).

The terms “inhibit” or “inhibition of” as used herein means to reduce bya measurable amount, or to prevent entirely.

The phrases “isolated” or “biologically pure” refer to material which issubstantially or essentially free from components which normallyaccompany the material as it is found in its native state. Thus,isolated peptides in accordance with the invention preferably do notcontain materials normally associated with the peptides in their in situenvironment. For example, a polynucleotide is said to be “isolated” whenit is substantially separated from contaminant polynucleotides thatcorrespond or are complementary to genes other than the AXL genes orthat encode polypeptides other than AXL gene product or fragmentsthereof. A skilled artisan can readily employ nucleic acid isolationprocedures to obtain an isolated AXL polynucleotide. A protein is saidto be “isolated,” for example, when physical, mechanical or chemicalmethods are employed to remove the AXL proteins from cellularconstituents that are normally associated with the protein. A skilledartisan can readily employ standard purification methods to obtain anisolated AXL protein. Alternatively, an isolated protein can be preparedby chemical means.

Suitable “labels” include radionuclides, enzymes, substrates, cofactors,inhibitors, fluorescent moieties, chemiluminescent moieties, magneticparticles, and the like. Patents teaching the use of such labels includeU.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437;4,275,149; and 4,366,241. In addition, the antibodies provided hereincan be useful as the antigen-binding component of fluorobodies. Seee.g., Zeytun et al., Nat. Biotechnol. 21:1473-79 (2003).

The term “mammal” refers to any organism classified as a mammal,including mice, rats, rabbits, dogs, cats, cows, horses and humans. Inone embodiment of the invention, the mammal is a mouse. In anotherembodiment of the invention, the mammal is a human.

The terms “metastatic cancer” and “metastatic disease” mean cancers thathave spread to regional lymph nodes or to distant sites, and are meantto include stage D disease under the AUA system and stage T×N×M+ underthe TNM system.

The term “modulator” or “test compound” or “drug candidate” orgrammatical equivalents as used herein describe any molecule, e.g.,protein, oligopeptide, small organic molecule, polysaccharide,polynucleotide, etc., to be tested for the capacity to directly orindirectly alter the cancer phenotype or the expression of a cancersequence, e.g., a nucleic acid or protein sequences, or effects ofcancer sequences (e.g., signaling, gene expression, protein interaction,etc.) In one aspect, a modulator will neutralize the effect of a cancerprotein of the invention. By “neutralize” is meant that an activity of aprotein is inhibited or blocked, along with the consequent effect on thecell. In another aspect, a modulator will neutralize the effect of agene, and its corresponding protein, of the invention by normalizinglevels of said protein. In preferred embodiments, modulators alterexpression profiles, or expression profile nucleic acids or proteinsprovided herein, or downstream effector pathways. In one embodiment, themodulator suppresses a cancer phenotype, e.g. to a normal tissuefingerprint. In another embodiment, a modulator induced a cancerphenotype. Generally, a plurality of assay mixtures is run in parallelwith different agent concentrations to obtain a differential response tothe various concentrations. Typically, one of these concentrationsserves as a negative control, i.e., at zero concentration or below thelevel of detection.

Modulators, drug candidates, or test compounds encompass numerouschemical classes, though typically they are organic molecules,preferably small organic compounds having a molecular weight of morethan 100 and less than about 2,500 Daltons. Preferred small moleculesare less than 2000, or less than 1500 or less than 1000 or less than 500D. Candidate agents comprise functional groups necessary for structuralinteraction with proteins, particularly hydrogen bonding, and typicallyinclude at least an amine, carbonyl, hydroxyl or carboxyl group,preferably at least two of the functional chemical groups. The candidateagents often comprise cyclical carbon or heterocyclic structures and/oraromatic or polyaromatic structures substituted with one or more of theabove functional groups. Modulators also comprise biomolecules such aspeptides, saccharides, fatty acids, steroids, purines, pyrimidines,derivatives, structural analogs or combinations thereof. Particularlypreferred are peptides. One class of modulators are peptides, forexample of from about five to about 35 amino acids, with from about fiveto about 20 amino acids being preferred, and from about 7 to about 15being particularly preferred. Preferably, the cancer modulatory proteinis soluble, includes a non-transmembrane region, and/or, has anN-terminal Cys to aid in solubility. In one embodiment, the C-terminusof the fragment is kept as a free acid and the N-terminus is a freeamine to aid in coupling, i.e., to cysteine. In one embodiment, a cancerprotein of the invention is conjugated to an immunogenic agent asdiscussed herein. In one embodiment, the cancer protein is conjugated toBSA. The peptides of the invention, e.g., of preferred lengths, can belinked to each other or to other amino acids to create a longerpeptide/protein. The modulatory peptides can be digests of naturallyoccurring proteins as is outlined above, random peptides, or “biased”random peptides. In a preferred embodiment, peptide/protein-basedmodulators are antibodies, and fragments thereof, as defined herein.

The term “monoclonal antibody”, as used herein, refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic epitope. In contrast, conventional(polyclonal) antibody preparations typically include a multitude ofantibodies directed against (or specific for) different epitopes. In oneembodiment, the polyclonal antibody contains a plurality of monoclonalantibodies with different epitope specificities, affinities, oravidities within a single antigen that contains multiple antigenicepitopes. The modifier “monoclonal” indicates the character of theantibody as being obtained from a substantially homogeneous populationof antibodies, and is not to be construed as requiring production of theantibody by any particular method. For example, the monoclonalantibodies to be used in accordance with the present invention may bemade by the hybridoma method first described by Kohler et al., Nature256: 495 (1975), or may be made by recombinant DNA methods (see, e.g.,U.S. Pat. No. 4,816,567). The “monoclonal antibodies” may also beisolated from phage antibody libraries using the techniques described inClackson et al., Nature 352: 624-628 (1991) and Marks et al., J. Mol.Biol. 222: 581-597 (1991), for example. These monoclonal antibodies willusually bind with at least a Kd of about 1 μM, more usually at leastabout 300 nM, typically at least about 30 nM, preferably at least about10 nM, more preferably at least about 3 nM or better, usually determinedby ELISA.

A “pharmaceutical excipient” comprises a material such as an adjuvant, acarrier, pH-adjusting and buffering agents, tonicity adjusting agents,wetting agents, preservative, and the like.

“Pharmaceutically acceptable” refers to a non-toxic, inert, and/orcomposition that is physiologically compatible with humans or othermammals.

The term “polynucleotide” means a polymeric form of nucleotides of atleast 10 bases or base pairs in length, either ribonucleotides ordeoxynucleotides or a modified form of either type of nucleotide, and ismeant to include single and double stranded forms of DNA and/or RNA. Inthe art, this term if often used interchangeably with “oligonucleotide”.A polynucleotide can comprise a nucleotide sequence disclosed hereinwherein thymidine (T), as shown for example in FIG. 1, can also beuracil (U); this definition pertains to the differences between thechemical structures of DNA and RNA, in particular the observation thatone of the four major bases in RNA is uracil (U) instead of thymidine(T).

The term “polypeptide” means a polymer of at least about 4, 5, 6, 7, or8 amino acids. Throughout the specification, standard three letter (See,Table III) or single letter designations for amino acids are used. Inthe art, this term is often used interchangeably with “peptide” or“protein”.

A “recombinant” DNA or RNA molecule is a DNA or RNA molecule that hasbeen subjected to molecular manipulation in vitro.

As used herein, the term “single-chain Fv” or “scFv” or “single chain”antibody refers to antibody fragments comprising the V_(H) and V_(L)domains of antibody, wherein these domains are present in a singlepolypeptide chain. Generally, the Fv polypeptide further comprises apolypeptide linker between the V_(H) and V_(L) domains which enables thesFv to form the desired structure for antigen binding. For a review ofsFv, see Pluckthun, THE PHARMACOLOGY OF MONOCLONAL ANTIBODIES, vol. 113,Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315 (1994).

As used herein, the terms “specific”, “specifically binds” and “bindsspecifically” refer to the selective binding of the antibody to thetarget antigen epitope. Antibodies can be tested for specificity ofbinding by comparing binding to appropriate antigen to binding toirrelevant antigen or antigen mixture under a given set of conditions.If the antibody binds to the appropriate antigen at least 2, 5, 7, andpreferably 10 times more than to irrelevant antigen or antigen mixturethen it is considered to be specific. In one embodiment, a specificantibody is one that only binds the AXL antigen, but does not bind tothe irrelevent antigen. In another embodiment, a specific antibody isone that binds human AXL antigen but does not bind a non-human AXLantigen with 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or greater amino acid homology with the AXL antigen. In anotherembodiment, a specific antibody is one that binds human AXL antigen andbinds murine AXL antigen, but with a higher degree of binding the humanantigen. In another embodiment, a specific antibody is one that bindshuman AXL antigen and binds primate AXL antigen, but with a higherdegree of binding the human antigen. In another embodiment, the specificantibody binds to human AXL antigen and any non-human AXL antigen, butwith a higher degree of binding the human antigen or any combinationthereof.

As used herein “to treat” or “therapeutic” and grammatically relatedterms, refer to any improvement of any consequence of disease, such asprolonged survival, less morbidity, and/or a lessening of side effectswhich are the byproducts of an alternative therapeutic modality; as isreadily appreciated in the art, full eradication of disease is apreferred but albeit not a requirement for a treatment act.

The term “variant” refers to a molecule that exhibits a variation from adescribed type or norm, such as a protein that has one or more differentamino acid residues in the corresponding position(s) of a specificallydescribed protein (e.g. the AXL protein shown in FIG. 1.) An analog isan example of a variant protein. Splice isoforms and single nucleotidespolymorphisms (SNPs) are further examples of variants.

The “AXL proteins” and/or “AXL related proteins” of the inventioninclude those specifically identified herein (see, FIG. 1), as well asallelic variants, conservative substitution variants, analogs andhomologs that can be isolated/generated and characterized without undueexperimentation following the methods outlined herein or readilyavailable in the art. Fusion proteins that combine parts of differentAXL proteins or fragments thereof, as well as fusion proteins of a AXLprotein and a heterologous polypeptide are also included. Such AXLproteins are collectively referred to as the AXL-related proteins, theproteins of the invention, or AXL. The term “AXL-related protein” refersto a polypeptide fragment or a AXL protein sequence of 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or morethan 25 amino acids; or, at least 30, 35, 40, 45, 50, 55, 60, 65, 70,80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150,155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 225, 250, 275, 280,290, 300, 325, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575,600, 625, 650, 675, 700, 725, 750, 775, 800, 810, 820, 830, 840, 841,842, 843, 844, 845, 850, 855, 860, 865, 870, 875, 880, 885, 890, 891,892, 893 or 894 or more amino acids.

II.) AXL Antibodies

Another aspect of the invention provides antibodies that bind toAXL-related proteins (See FIG. 1). In one embodiment, the antibody thatbinds to AXL-related proteins is an antibody that specifically binds toAXL protein comprising amino acid sequence of SEQ ID NO.: 2. Theantibody that specifically binds to AXL protein comprising amino acidsequence of SEQ ID NO.: 2 includes antibodies that can bind to otherAXL-related proteins. For example, antibodies that bind AXL proteincomprising amino acid sequence of SEQ ID NO.: 2 can bind AXL-relatedproteins such as AXL variants and the homologs or analogs thereof.

AXL antibodies of the invention are particularly useful in cancer (see,e.g., Table I) prognostic assays, imaging, diagnostic, and therapeuticmethodologies. Similarly, such antibodies are useful in the treatment,and/or prognosis of sarcoma, pancreatic, melanoma, ovarian, or lung andother cancers, to the extent AXL is also expressed or overexpressed inthese other cancers. Moreover, intracellularly expressed antibodies(e.g., single chain antibodies) are therapeutically useful in treatingcancers in which the expression of AXL is involved, such as advanced ormetastatic sarcoma, pancreatic, melanoma, ovarian, or lung cancers orother advanced or metastatic cancers.

Various methods for the preparation of antibodies, specificallymonoclonal antibodies, are well known in the art. For example,antibodies can be prepared by immunizing a suitable mammalian host usinga AXL-related protein, peptide, or fragment, in isolated orimmunoconjugated form (Antibodies: A Laboratory Manual, CSH Press, Eds.,Harlow, and Lane (1988); Harlow, Antibodies, Cold Spring Harbor Press,NY (1989)). In addition, fusion proteins of AXL can also be used, suchas a AXL GST-fusion protein. In a particular embodiment, a GST fusionprotein comprising all or most of the amino acid sequence of FIG. 1 isproduced, and then used as an immunogen to generate appropriateantibodies. In another embodiment, a AXL-related protein is synthesizedand used as an immunogen.

In addition, naked DNA immunization techniques known in the art are used(with or without purified AXL-related protein or AXL expressing cells)to generate an immune response to the encoded immunogen (for review, seeDonnelly et al., 1997, Ann. Rev. Immunol. 15: 617-648).

The amino acid sequence of a AXL protein as shown in FIG. 1 can beanalyzed to select specific regions of the AXL protein for generatingantibodies. For example, hydrophobicity and hydrophilicity analyses of aAXL amino acid sequence are used to identify hydrophilic regions in theAXL structure. Regions of a AXL protein that show immunogenic structure,as well as other regions and domains, can readily be identified usingvarious other methods known in the art, such as Chou-Fasman,Garnier-Robson, Kyte-Doolittle, Eisenberg, Karplus-Schultz orJameson-Wolf analysis. Hydrophilicity profiles can be generated usingthe method of Hopp, T. P. and Woods, K. R., 1981, Proc. Natl. Acad. Sci.U.S.A. 78:3824-3828. Hydropathicity profiles can be generated using themethod of Kyte, J. and Doolittle, R. F., 1982, J. Mol. Biol.157:105-132. Percent (%) Accessible Residues profiles can be generatedusing the method of Janin J., 1979, Nature 277:491-492. AverageFlexibility profiles can be generated using the method of Bhaskaran R.,Ponnuswamy P. K., 1988, Int. J. Pept. Protein Res. 32:242-255. Beta-turnprofiles can be generated using the method of Deleage, G., Roux B.,1987, Protein Engineering 1:289-294. Thus, each region identified by anyof these programs or methods is within the scope of the presentinvention. Preferred methods for the generation of AXL antibodies arefurther illustrated by way of the examples provided herein. Methods forpreparing a protein or polypeptide for use as an immunogen are wellknown in the art. Also well known in the art are methods for preparingimmunogenic conjugates of a protein with a carrier, such as BSA, KLH orother carrier protein. In some circumstances, direct conjugation using,for example, carbodiimide reagents are used; in other instances linkingreagents such as those supplied by Pierce Chemical Co., Rockford, Ill.,are effective. Administration of a AXL immunogen is often conducted byinjection over a suitable time period and with use of a suitableadjuvant, as is understood in the art. During the immunization schedule,titers of antibodies can be taken to determine adequacy of antibodyformation.

AXL monoclonal antibodies can be produced by various means well known inthe art. For example, immortalized cell lines that secrete a desiredmonoclonal antibody are prepared using the standard hybridoma technologyof Kohler and Milstein or modifications that immortalizeantibody-producing B cells, as is generally known. Immortalized celllines that secrete the desired antibodies are screened by immunoassay inwhich the antigen is a AXL-related protein. When the appropriateimmortalized cell culture is identified, the cells can be expanded andantibodies produced either from in vitro cultures or from ascites fluid.

The antibodies or fragments of the invention can also be produced byrecombinant means. Regions that bind specifically to the desired regionsof a AXL protein can also be produced in the context of chimeric orcomplementarity-determining region (CDR) grafted antibodies of multiplespecies origin. Humanized or human AXL antibodies can also be produced,and are preferred for use in therapeutic contexts. Methods forhumanizing murine and other non-human antibodies, by substituting one ormore of the non-human antibody CDRs for corresponding human antibodysequences, are well known (see for example, Jones et al., 1986, Nature321: 522-525; Riechmann et al., 1988, Nature 332: 323-327; Verhoeyen etal., 1988, Science 239: 1534-1536). See also, Carter et al., 1993, Proc.Natl. Acad. Sci. USA 89: 4285 and Sims et al., 1993, J. Immunol. 151:2296.

In a preferred embodiment, human monoclonal antibodies of the inventioncan be prepared using VelocImmune mice into which genomic sequencesbearing endogenous mouse variable segments at the immunoglobulin heavychain (VH, DH, and JH segments) and/or kappa light chain (VK and JK)loci have been replaced, in whole or in part, with human genomicsequences bearing unrearranged germline variable segments of the humanimmunoglobulin heavy chain (VH, DH, and JH) and/or kappa light chain (VKand JK) loci (Regeneron, Tarrytown, N.Y.). See, for example, U.S. Pat.Nos. 6,586,251, 6,596,541, 7,105,348, 6,528,313, 6,638,768, and6,528,314.

In addition, human antibodies of the invention can be generated usingthe HuMAb mouse (Medarex, Inc.) which contains human immunoglobulin geneminiloci that encode unrearranged human heavy (mu and gamma) and kappalight chain immunoglobulin sequences, together with targeted mutationsthat inactivate the endogenous mu and kappa chain loci (see e.g.,Lonberg, et al. (1994) Nature 368(6474): 856-859).

In another embodiment, fully human antibodies of the invention can beraised using a mouse that carries human immunoglobulin sequences ontransgenes and transchomosomes, such as a mouse that carries a humanheavy chain transgene and a human light chain transchromosome. Suchmice, referred to herein as “KM mice”, such mice are described inTomizuka et al. (2000) Proc. Natl. Acad. Sci. USA 97:722-727 and PCTPublication WO 02/43478 to Tomizuka, et al.

Human monoclonal antibodies of the invention can also be prepared usingphage display methods for screening libraries of human immunoglobulingenes. Such phage display methods for isolating human antibodies areestablished in the art. See for example: U.S. Pat. Nos. 5,223,409;5,403,484; and 5,571,698 to Ladner et al.; U.S. Pat. Nos. 5,427,908 and5,580,717 to Dower et al.; U.S. Pat. Nos. 5,969,108 and 6,172,197 toMcCafferty et al.; and U.S. Pat. Nos. 5,885,793; 6,521,404; 6,544,731;6,555,313; 6,582,915 and 6,593,081 to Griffiths et al.

Human monoclonal antibodies of the invention can also be prepared usingSCID mice into which human immune cells have been reconstituted suchthat a human antibody response can be generated upon immunization. Suchmice are described in, for example, U.S. Pat. Nos. 5,476,996 and5,698,767 to Wilson et al.

Additionally, human antibodes of the present invention can be made withtechniques using transgenic mice, inactivated for antibody production,engineered with human heavy and light chains loci referred to asXenomouse (Amgen Fremont, Inc.). An exemplary description of preparingtransgenic mice that produce human antibodies can be found in U.S. Pat.No. 6,657,103. See, also, U.S. Pat. Nos. 5,569,825; 5,625,126;5,633,425; 5,661,016; and 5,545,806; and Mendez, et. al. NatureGenetics, 15: 146-156 (1998); Kellerman, S. A. & Green, L. L., Curr.Opin. Biotechnol 13, 593-597 (2002).

In one embodiment, an AXL MAbs of the invention comprises heavy andlight chain CDRs of an antibody designated V77-2a37.1, whereby the heavychain variable region is set forth in FIG. 3A as SEQ ID NO: 8 andwhereby the light chain variable region is set forth in FIG. 3B as SEQID NO: 10. In one aspect of invention, an AXL MAbs of the inventioncomprises a heavy chain comprising CDR-H1 consisting of residues 31-37(SEQ ID NO: 20), CDR-H2 consisting of residues 50-65 (SEQ ID NO: 22) andCDR-H3 consisting of residues 95-102 (SEQ ID NO: 23) and a light chaincomprising CDR-L1 consisting of residues 24-34 (SEQ ID NO: 11), a CDR-L2consisting of residues 50-56 (SEQ ID NO: 12) and CDR-L3 consisting ofresidues 89-97 (SEQ ID NO: 13). In a preferred embodiment, an AXL MAbsof the invention comprises heavy and light chain variable regions of anantibody designated V77-2a37.1, whereby the heavy chain variable regionis set forth in FIG. 3A as SEQ ID NO: 8 and whereby the light chainvariable region is set forth in FIG. 3B as SEQ ID NO: 10. The MAbs ofthe inventions comprise heavy and light variable regions comprisingamino acid sequences that are homologous to the amino acid sequences ofthe heavy and light chain variable regions of V77-2a37.1, and whereinthe antibodies retain the desired functional properties of the AXL MAbsof the invention. It should be noted, as the constant region of theantibody of the invention, any subclass of constant region can bechosen. In one embodiment, human IgG2 constant region as the heavy chainconstant region and human Ig kappa constant region as the light chainconstant region can be used. In other embodiment, mouse IgG1 constantregion as the heavy chain constant region and mouse Ig kappa constantregion as the light chain constant region can be used.

Engineered antibodies of the invention include those in whichmodifications have been made to framework residues within V_(H) and/orV_(L) (e.g. to improve the properties of the antibody). Typically suchframework modifications are made to decrease the immunogenicity of theantibody. For example, one approach is to “backmutate” one or moreframework residues to the corresponding germline sequence. Morespecifically, an antibody that has undergone somatic mutation maycontain framework residues that differ from the germline sequence fromwhich the antibody is derived. Such residues can be identified bycomparing the antibody framework sequences to the germline sequencesfrom which the antibody is derived. To return the framework regionsequences to their germline configuration, the somatic mutations can be“backmutated” to the germline sequence by, for example, site-directedmutagenesis or PCR-mediated mutagenesis (e.g., “backmutated” fromleucine to methionine). Such “backmutated” antibodies are also intendedto be encompassed by the invention.

Another type of framework modification involves mutating one or moreresidues within the framework region, or even within one or more CDRregions, to remove T-cell epitopes to thereby reduce the potentialimmunogenicity of the antibody. This approach is also referred to as“deimmunization” and is described in further detail in U.S. PatentPublication No. 2003/0153043 by Carr et al.

In addition or alternative to modifications made within the framework orCDR regions, antibodies of the invention may be engineered to includemodifications within the Fc region, typically to alter one or morefunctional properties of the antibody, such as serum half-life,complement fixation, Fc receptor binding, and/or antigen-dependentcellular cytotoxicity. Furthermore, a AXL MAb of the invention may bechemically modified (e.g., one or more chemical moieties can be attachedto the antibody) or be modified to alter its glycosylation, again toalter one or more functional properties of the MAb. Each of theseembodiments is described in further detail below.

In one embodiment, the hinge region of CH1 is modified such that thenumber of cysteine residues in the hinge region is altered, e.g.,increased or decreased. This approach is described further in U.S. Pat.No. 5,677,425 by Bodmer et al. The number of cysteine residues in thehinge region of CH1 is altered to, for example, facilitate assembly ofthe light and heavy chains or to increase or decrease the stability ofthe AXL MAb.

In another embodiment, the Fc hinge region of an antibody is mutated todecrease the biological half life of the AXL MAb. More specifically, oneor more amino acid mutations are introduced into the CH2-CH3 domaininterface region of the Fc-hinge fragment such that the antibody hasimpaired Staphylococcyl protein A (SpA) binding relative to nativeFc-hinge domain SpA binding. This approach is described in furtherdetail in U.S. Pat. No. 6,165,745 by Ward et al.

In another embodiment, the AXL MAb is modified to increase itsbiological half life. Various approaches are possible. For example,mutations can be introduced as described in U.S. Pat. No. 6,277,375 toWard. Alternatively, to increase the biological half life, the antibodycan be altered within the CH1 or CL region to contain a salvage receptorbinding epitope taken from two loops of a CH2 domain of an Fc region ofan IgG, as described in U.S. Pat. Nos. 5,869,046 and 6,121,022 by Prestaet al.

In yet other embodiments, the Fc region is altered by replacing at leastone amino acid residue with a different amino acid residue to alter theeffector function(s) of the AXL MAb. For example, one or more aminoacids selected from amino acid specific residues can be replaced with adifferent amino acid residue such that the antibody has an alteredaffinity for an effector ligand but retains the antigen-binding abilityof the parent antibody. The effector ligand to which affinity is alteredcan be, for example, an Fc receptor or the Cl component of complement.This approach is described in further detail in U.S. Pat. Nos. 5,624,821and 5,648,260, both by Winter et al.

Reactivity of AXL antibodies with a AXL-related protein can beestablished by a number of well known means, including Western blot,immunoprecipitation, ELISA, and FACS analyses using, as appropriate,AXL-related proteins, AXL-expressing cells or extracts thereof. A AXLantibody or fragment thereof can be labeled with a detectable marker orconjugated to a second molecule. Suitable detectable markers include,but are not limited to, a radioisotope, a fluorescent compound, abioluminescent compound, chemiluminescent compound, a metal chelator oran enzyme. Further, bi-specific antibodies specific for two or more AXLepitopes are generated using methods generally known in the art.Homodimeric antibodies can also be generated by cross-linking techniquesknown in the art (e.g., Wolff et al., Cancer Res. 53: 2560-2565).

In yet another preferred embodiment, the AXL MAb of the invention is anantibody comprising heavy and light chain of an antibody designatedV77-2a37.1. In a preferred embodiment, V77-2a37.1 is conjugated to animaging agent. In a further preferred embodiment, V77-2a37.1 is used inconjunction with a small molecule compound as a companion diagnostic(CDx).

III.) Diagnosis of Cancer(s) Expressing AXL

The AXL MAb of the invention can be used in the detection method of thepresence of cancer in a subject, or in a diagnosis method of cancer in asubject. The example of cancer in the detection method or diagnosismethod includes sarcoma, pancreatic cancer, melanoma, ovarian cancer,and lung cancer. In addition, the AXL MAb of the invention can be usedin the detection method of AXL expression in sample obtained from asubject such as cancer patients. It is known that AXL expression incancer cells relates to resistance to EGFR inhibitor (e.g, Erlotinib) incancer patient which has undergone treatment by EGFR inhibitor.Therefore, the combination of AXL kinase inhibitor and EGFR inhibitorcan be used for diminishment of EGFR inhibitor resistance andsuppression of cancer growth. The AXL MAb of the invention can be usedin a method for identifying a cancer patient who is positive for AXLexpression, and therefore is subject to treatment by AXL kinaseinhibitor and/or EGFR inhibitor. In one embodiment, the cancer isselected from the group consisting of sarcoma, pancreatic cancer,melanoma, ovarian cancer, and lung cancer. In one embodiment, thepatient has undergone treatment by EGFR inhibitor and the cancer isresistant to EGFR inhibitor resistance.

The detection method comprises a step of detecting the existence of AXLprotein in a sample obtained from a subject. As the sample obtained froma subject, substances collected from a subject (samples separated from abiological body), specifically, any type of collected tissues, bodyfluids (preferably blood), bronchoalveolar lavages, samples havingundergone biopsy, cancer cells in urine, and sputum samples are used. Inone preferable embodiment, biopsy samples collected from the affectedsite of organ (e.g., lung) of a subject can be used. It is also possibleto use a specimen (FFPE) obtained by fixing the sample by using formalinand stabilizing the sample by embedding it in paraffin. Moreover, anFFPE slice obtained by cutting the FFPE into a thin slice may be used.If the FFPE slice is used, it is possible to directly detect AXL proteinexisting in the slice.

The step of detecting AXL protein can be conducted by using one of themethods known in the skilled in the art. For example, the detection maybe performed by a method as a combination of immunoassay and enzymaticactivity assay in which a solubilization solution derived from a sampleobtained from a test subject (for example, a cancer tissue or a cancercell obtained from a test subject) is prepared, and AXL proteincontained in the solution is combined with AXL antibody of theinvention. Furthermore, the detection may be performed by animmunohistostaining technique in which AXL protein contained in a sample(for example, an FFPE fragment) obtained from a test subject which hasundergone pretreatment (for example, removal of paraffin) as appropriateis combined with the AXL antibody of the invention. Examples of thesetechniques include techniques such as enzymatic immunoassay, doubleantibody sandwich ELISA, fluorescence immunoassay, radioimmunoassay,Western blotting, and immunohistostaining.

In addition to the above embodiments, the below paragraphs describeadditional antibodies, antigen binding fragments, and uses and methodsusing the same:

Described herein, e.g. in this first paragraph, are antibodies andantigen binding fragments of such antibodies that bind AXL protein,suitably human AXL protein. In certain embodiments, the antibodies andantigen binding fragments specifically bind AXL protein, suitably humanAXL protein. Such antibodies comprise the complementary determiningregions consisting of the amino acid sequences as set forth in the SEQID NOs shown in Table VII, as identified by the Kabat, Chothia, andContact schemes. Thus, in one embodiment is an antibody or antigenbinding fragment comprising a CDR-L1 consisting of the amino acidsequence set forth in SEQ ID NO:11, a CDR-L2 consisting of the aminoacid sequence set forth in SEQ ID NO:12, a CDR-L3 consisting of theamino acid sequence set forth in SEQ ID NO:13, a CDR-H1 consisting ofthe amino acid sequence set forth in SEQ ID NO: 20, a CDR-H2 consistingof the amino acid sequence set forth in SEQ ID NO: 22, and a CDR-H3consisting of the amino acid sequence set forth in SEQ ID NO: 23. In analternative embodiment is an antibody or antigen binding fragmentcomprising a CDR-L1 consisting of the amino acid sequence set forth inSEQ ID NO:14, a CDR-L2 consisting of the amino acid sequence set forthin SEQ ID NO:15, a CDR-L3 consisting of the amino acid sequence setforth in SEQ ID NO:16, a CDR-H1 consisting of the amino acid sequenceset forth in SEQ ID NO: 25, a CDR-H2 consisting of the amino acidsequence set forth in SEQ ID NO: 26, and a CDR-H3 consisting of theamino acid sequence set forth in SEQ ID NO: 27. In yet anotheralternative embodiment is an antibody or antigen binding fragmentcomprising a CDR-L1 consisting of the amino acid sequence set forth inSEQ ID NO:17, a CDR-L2 consisting of the amino acid sequence set forthin SEQ ID NO:18, a CDR-L3 consisting of the amino acid sequence setforth in SEQ ID NO:19, a CDR-H1 consisting of the amino acid sequenceset forth in SEQ ID NO: 28, a CDR-H2 consisting of the amino acidsequence set forth in SEQ ID NO: 30, and a CDR-H3 consisting of theamino acid sequence set forth in SEQ ID NO: 31.

Another embodiment is an antibody or antigen binding fragment accordingto the preceding paragraph, wherein the antibody or antigen bindingfragment comprises a heavy chain variable region consisting of the aminoacid sequence set forth in SEQ ID NO: 8 and a light chain variableregion consisting of the amino acid set forth in SEQ ID NO: 10.

Another embodiment, disclosed herein, e.g., in this third paragraph, arefull length antibodies. Thus, one embodiment is an antibody or antigenbinding fragment according to any one of the preceding paragraphs,wherein the antibody comprises a heavy chain consisting of the aminoacid sequence set forth in SEQ ID NO: 7 and a light chain consisting ofthe amino acid sequence set forth in SEQ ID NO: 9.

Also disclosed herein, e.g. in this fourth paragraph, are antibodiesthat bind AXL protein, suitably human AXL protein, that have substantialsequence identity to those described in the preceding paragraphs. Thus,one embodiment of this paragraph is an antibody or antigen bindingfragment thereof that has 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,or 99% to an antibody or antigen binding fragment of any of thepreceding paragraphs. In a further embodiment, the antibody or antigenbinding fragment thereof has 95% or greater sequence identity to theantibody or antigen binding fragment according to any of the precedingparagraphs. In further embodiments, the antibodies or antigen bindingfragments in the instant paragraph have amino acid changes that do notsubstantially change the binding affinity relative to the originalunmodified sequence. In certain embodiments such antibodies or antigenbinding fragments have about the same binding affinity, that is theantibody or antigen binding fragment of this instant paragraphspecifically binds AXL protein with less than 1%, 2%, 3%, 4%, 5%, 6%,7%, 8%, 10%, 11%, 12%, 13%, or 14%, or 15% change as compared to thebinding affinity of the original antibody or antigen binding fragmentaccording to any one of the preceding paragraphs. A further and suitableembodiment is an antibody or antigen binding fragment that has 99%sequence identity to the original antibody or fragment according to anyone of the preceding paragraphs, and wherein any amino acid changes arenot to a residue in any CDR regions, wherein the CDR regions aredetermined using Kabat numbering. In an alternative embodiment theantibody or antigen binding fragment has 99% sequence identity to theoriginal antibody or fragment in the preceding paragraphs, and whereinany amino acid changes are not to a residue in any CDR regions, whereinthe CDR regions are determined by Chothia numbering. In an alternativeembodiment the antibody or antigen binding fragment has 99% sequenceidentity to the original antibody or fragment in the precedingparagraphs, and wherein any amino acid changes are not to a residue inany CDR regions, wherein the CDR regions are determined by the Contactmethod. In further embodiments, the antibody or antigen binding fragmentwith 99% sequence identity to the original antibody or antigen bindingfragment of the preceding paragraph has about the same binding affinityas the original antibody or antigen binding fragment.

Another embodiment, as disclosed herein, e.g. in this fifth paragraph isthe antibody or antigen binding fragment thereof according to any of thepreceding paragraphs, wherein the fragment is an Fab, F(ab′)2, Fv orscFv fragment. In certain embodiments an antigen binding fragment ofthis paragraph or any of the preceding paragraphs can be fused to aheterologous Fc region or a heterologous constant region resulting in achimeric antibody.

Another embodiment, as disclosed herein, e.g. in this sixth paragraph,is the antibody or antigen binding fragment according to any of thepreceding paragraphs, wherein the antibody or antigen binding fragmentis a fully human antibody.

Another embodiment, as disclosed herein, e.g. in this seventh paragraph,is the antibody or antigen binding fragment according to any of thepreceding paragraphs, wherein the antibody is an antibody designatedV77-2a37.1.

Another embodiment, as disclosed herein, e.g. in this eighth paragraph,is one or more polynucleotides encoding the antibody or antigen bindingfragment according to any one of the preceding paragraphs. In oneembodiment, the heavy chain variable region or the heavy chain isencoded by one polynucleotide, and the light chain variable region orthe light chain is encoded on a second polynucleotide. In a furtherembodiment, the one or more polynucleotides is a vector.

In further embodiments disclosed herein, e.g. in this ninth paragraph,are host cells comprising the one or more polynucleotides of thepreceding paragraph. In one embodiment is a host cell comprising onevector comprising the heavy chain variable region or the heavy chain ofan antibody or antigen binding fragment according to any one ofparagraphs one to seven, and another vector comprising the light chainvariable region or the light chain of an antibody or antigen bindingfragment according to any one of paragraphs one to seven.

In a further embodiment as disclosed herein, e.g. in this tenthparagraph, is an antibody or antigen binding fragment thereof producedby the host cell of the preceding paragraph. In a further embodiment,the antibody or antigen binding fragment so produced is further isolatedby one or more techniques known in the art, including ion exchangechromatography, HPLC, size exclusion chromatography, SDS PAGE, affinitychromatography, and the like. In a further embodiment, the antibody orantigen binding fragment is about 90% or more pure. Another embodimentas disclosed herein, e.g. in this tenth paragraph, is an antibody orantigen binding fragment there of that competes for binding with theantibody or antigen binding fragment according to any one of paragraphsone to seven. In a further embodiment, the antibody that competes forbinding in an ELISA assay comprising a multi-well plate coated with AXLprotein, suitably human protein.

Another embodiment is the antibody or antigen binding fragment accordingto any one of paragraphs one to seven, or paragraph 10, wherein theantibody or antigen binding fragment is recombinantly produced.

Another embodiment is the antibody or antigen binding fragment accordingto any one of paragraphs one to seven, or paragraph 10, wherein theantibody or antigen binding fragment is conjugated to an imaging agent.In an alternative embodiment, the antibody or antigen binding fragmentaccording to any of the preceding paragraphs is for use as an imagingagent. In a further embodiment in an antibody or antigen bindingfragment of any one of the preceding paragraphs for use as in vivoimaging agent. In an alternative embodiment, antibody or antigen bindingfragment of any one of the preceding paragraphs for use an ex vivoimaging agent, suitably for use in a detection assay; such assaysinclude, but are not limited to, immunohistochemistry,immunocytochemistry, immunoassays, ELISAs (including double antibodysandwich ELISAs), and Western blotting. A further embodiment is anantibody or antigen binding fragment according to any of the precedingparagraphs, wherein the antibody or antigen binding fragment isconjugated, directly or indirectly, to one or more detection reagents.The assays may be qualitative or quantitative and employ any number ofdetection reagents, including but not limited to, radioactivity,fluorescence, chemiluminescence and may also involve enzyme linkeddetection using any of the preceding reagents.

In a different embodiment is the antibody or antigen binding fragmentaccording to any one of paragraphs one to seven, or paragraph 10, foruse as a companion diagnostic (CDx) to screen one or more patients forthe diagnosis and/or treatment and/or management of cancer, suitablysarcoma, pancreatic cancer, melanoma, ovarian cancer, and lung cancer.

In a different embodiment is a method of detecting AXL expression in oneor more tumor cells in a subject, comprising a step of contacting theantibody or antigen binding fragment according to any one of paragraphsone to seven, or paragraph 10, with a sample of one or more cellsobtained from the subject. In a further embodiment, the subject is ahuman. In a further embodiment, the human in need of diagnosis and/ortreatment of cancer. In a further embodiment, the one or more tumorcells are suitably cells of a solid organ tumor, suitably pancreatic,ovarian, lung, and skin. In an alternative embodiment, the one or moretumor cells are soft tissue cells of mesenchymal origin. In anotherembodiment, the one or more cells are selected from the group consistingof sarcoma cells, pancreatic tumor cells, melanoma cells, ovarian tumorcells, and lung tumor cells. In a further embodiment, the subject,suitably a human, was previously treated with an EGFR inhibitor. In afurther embodiment, the subject, suitably a human, previously treatedwith an EGFR inhibitor was determined to have a cancer that did notsubstantially respond to an EGFR inhibitor, that is, an EGFR resistantcancer.

In a different embodiment is a method for identifying a subject,suitably a human, having one or more cancer cells which are positive forAXL expression, comprising contacting the antibody or antigen bindingfragment according to any one of paragraphs one to seven, or paragraph10, with a sample of one or more cells obtained from the subject,suitably a human, and detecting binding of the antibody or antigenbinding fragment to AXL. In a further embodiment, the subject, suitablya human, was previously treated with an EGFR inhibitor. In a furtherembodiment, the subject, suitably a human, previously treated with anEGFR inhibitor was determined to have an EGFR resistant cancer.

In yet a different embodiment is a method of diagnosing cancer in asubject, suitably a human, the method comprising contacting the antibodyor antigen binding fragment according to any one of paragraphs one toseven, or paragraph 10, with a sample of one or more cells obtained fromthe subject, suitably a human, detecting binding of the antibody orantigen binding fragment to AXL, and determining an increased level ofAXL as compared to a control sample of one or more cells that are fromnormal, that is non-tumor or noncancerous, cells, and thereby diagnosingcancer. In a further embodiment, the subject, suitably a human, waspreviously treated with an EGFR inhibitor. In a further embodiment, thesubject, suitably a human, previously treated with an EGFR inhibitor wasdetermined to have an EGFR resistant cancer.

IV.) Treatment of Cancer(s) Expressing AXL

The identification of AXL as a protein that is normally expressed in arestricted set of tissues, but which is also expressed in cancers suchas those listed in Table I, opens a number of therapeutic and diagnosticapproaches to the treatment of such cancers.

Of note, targeted antitumor therapies have been useful even when thetargeted protein is expressed on normal tissues, even vital normal organtissues. A vital organ is one that is necessary to sustain life, such asthe heart or colon. A non-vital organ is one that can be removedwhereupon the individual is still able to survive. Examples of non-vitalorgans are ovary, breast, and prostate.

Expression of a target protein in normal tissue, even vital normaltissue, does not defeat the utility of a targeting agent for the proteinas a therapeutic for certain tumors in which the protein is alsooverexpressed. For example, expression in vital organs is not in and ofitself detrimental. In addition, organs regarded as dispensible, such asthe prostate and ovary, can be removed without affecting mortality.Finally, some vital organs are not affected by normal organ expressionbecause of an immunoprivilege. Immunoprivileged organs are organs thatare protected from blood by a blood-organ barrier and thus are notaccessible to immunotherapy. Examples of immunoprivileged organs are thebrain and testis.

Accordingly, therapeutic approaches that inhibit the activity of a AXLprotein are useful for patients suffering from a cancer that expressesAXL. These therapeutic approaches generally fall into three classes. Thefirst class modulates AXL function as it relates to tumor cell growthleading to inhibition or retardation of tumor cell growth or inducingits killing. The second class comprises various methods for inhibitingthe binding or association of a AXL protein with its binding partner orwith other proteins. The third class comprises a variety of methods forinhibiting the transcription of a AXL gene or translation of AXL mRNA.

Accordingly, Cancer patients can be evaluated for the presence and levelof AXL expression, preferably using immunohistochemical assessments oftumor tissue, quantitative AXL imaging, or other techniques thatreliably indicate the presence and degree of AXL expression.Immunohistochemical analysis of tumor biopsies or surgical specimens ispreferred for this purpose. Methods for immunohistochemical analysis oftumor tissues are well known in the art.

V.) AXL as a Target for Antibody-based Therapy

AXL is an attractive target for antibody-based therapeutic strategies. Anumber of antibody strategies are known in the art for targeting bothextracellular and intracellular molecules (see, e.g., complement andADCC mediated killing as well as the use of intrabodies). Because AXL isexpressed by cancer cells of various lineages relative to correspondingnormal cells, systemic administration of AXL-immunoreactive compositionsare prepared that exhibit excellent sensitivity without toxic,non-specific and/or non-target effects caused by binding of theimmunoreactive composition to non-target organs and tissues. Antibodiesspecifically reactive with domains of AXL are useful to treatAXL-expressing cancers systemically, preferably as antibody drugconjugates (i.e. ADCs) wherein the conjugate is with an imagin agent,toxin or therapeutic agent.

Those skilled in the art understand that antibodies can be used tospecifically target and bind immunogenic molecules such as animmunogenic region of a AXL sequence shown in FIG. 1. In addition,skilled artisans understand that it is routine to conjugate antibodiesto cytotoxic agents (see, e.g., Slevers et al. Blood 93:11 3678-3684(Jun. 1, 1999)). When cytotoxic and/or therapeutic agents are delivereddirectly to cells, such as by conjugating them to antibodies specificfor a molecule expressed by that cell (e.g. AXL), the cytotoxic agentwill exert its known biological effect (i.e. cytotoxicity) on thosecells.

A wide variety of compositions and methods for using antibody-cytotoxicagent conjugates to kill cells are known in the art. In the context ofcancers, typical methods entail administering to an mammal having atumor a biologically effective amount of a conjugate comprising aselected cytotoxic and/or therapeutic agent linked to a targeting agent(e.g. a AXL MAb, preferably V77-2a37.1) that binds to an antigen (e.g.AXL) expressed, accessible to binding or localized on the cell surfaces.A typical embodiment is a method of delivering a cytotoxic and/ortherapeutic agent to a cell expressing AXL, comprising conjugating thecytotoxic agent to an antibody that immunospecifically binds to a AXLepitope, and, exposing the cell to the antibody drug conjugate (ADC).Another illustrative embodiment is a method of treating an individualsuspected of suffering from metastasized cancer, comprising a step ofadministering parenterally to said individual a pharmaceuticalcomposition comprising a therapeutically effective amount of an antibodyconjugated to a cytotoxic and/or therapeutic agent.

Cancer immunotherapy using AXL antibodies can be done in accordance withvarious approaches that have been successfully employed in the treatmentof other types of cancer, including but not limited to colon cancer(Arlen et al., 1998, Crit. Rev. Immunol. 18:133-138), multiple myeloma(Ozaki et al., 1997, Blood 90:3179-3186, Tsunenari et al., 1997, Blood90:2437-2444), gastric cancer (Kasprzyk et al., 1992, Cancer Res.52:2771-2776), B-cell lymphoma (Funakoshi et al., 1996, J. Immunother.Emphasis Tumor Immunol. 19:93-101), leukemia (Zhong et al., 1996, Leuk.Res. 20:581-589), colorectal cancer (Moun et al., 1994, Cancer Res.54:6160-6166; Velders et al., 1995, Cancer Res. 55:4398-4403), andbreast cancer (Shepard et al., 1991, J. Clin. Immunol. 11:117-127). Sometherapeutic approaches involve conjugation of naked antibody to a toxinor radioisotope, such as the conjugation of Y⁹¹ or I¹³¹ to anti-CD20antibodies (e.g., Zevalin™, IDEC Pharmaceuticals Corp. or Bexxar™,Coulter Pharmaceuticals) respectively, while others involveco-administration of antibodies and other therapeutic agents, such asHerceptin™ (trastuzu MAb) with paclitaxel (Genentech, Inc.). In apreferred embodiment, the antibodies will be conjugated an imagingagent. In a further preferred embodiment, the MAbs will be used as acompanion diagnostic in conjunction with another compound for thetreatment of disease.

Although AXL antibody therapy is useful for all stages of cancer,antibody therapy can be particularly appropriate in advanced ormetastatic cancers. Treatment with the antibody therapy of the inventionis indicated for patients who have received one or more rounds ofchemotherapy. Alternatively, antibody therapy of the invention iscombined with a chemotherapeutic or radiation regimen for patients whohave not received chemotherapeutic treatment. Additionally, antibodytherapy can enable the use of reduced dosages of concomitantchemotherapy, particularly for patients who do not tolerate the toxicityof the chemotherapeutic agent very well. Fan et al. (Cancer Res.53:4637-4642, 1993), Prewett et al. (International J. of Onco.9:217-224, 1996), and Hancock et al. (Cancer Res. 51:4575-4580, 1991)describe the use of various antibodies together with chemotherapeuticagents.

AXL monoclonal antibodies that treat the cancers set forth in Table Iinclude those that initiate a potent immune response against the tumoror those that are directly cytotoxic. In this regard, AXL monoclonalantibodies (MAbs) can elicit tumor cell lysis by eithercomplement-mediated or antibody-dependent cell cytotoxicity (ADCC)mechanisms, both of which require an intact Fc portion of theimmunoglobulin molecule for interaction with effector cell Fc receptorsites on complement proteins. In addition, AXL MAbs that exert a directbiological effect on tumor growth are useful to treat cancers thatexpress AXL. Mechanisms by which directly cytotoxic MAbs act include:inhibition of cell growth, modulation of cellular differentiation,modulation of tumor angiogenesis factor profiles, and the induction ofapoptosis. The mechanism(s) by which a particular AXL MAb exerts ananti-tumor effect is evaluated using any number of in vitro assays thatevaluate cell death such as ADCC, complement-mediated cell lysis, and soforth, as is generally known in the art.

Accordingly, preferred monoclonal antibodies used in the therapeuticmethods of the invention are those that are either fully human and thatbind specifically to the target AXL antigen with high affinity.

VI.) AXL ADC Cocktails

Therapeutic methods of the invention contemplate the administration ofsingle AXL ADCs as well as combinations, or cocktails, of different MAbs(i.e. AXL MAbs or Mabs that bind another protein). Such MAb cocktailscan have certain advantages inasmuch as they contain MAbs that targetdifferent epitopes, exploit different effector mechanisms or combinedirectly cytotoxic MAbs with MAbs that rely on immune effectorfunctionality. Such MAbs in combination can exhibit synergistictherapeutic effects. In addition, AXL MAbs can be administeredconcomitantly with other therapeutic modalities, including but notlimited to various chemotherapeutic and biologic agents,androgen-blockers, immune modulators (e.g., IL-2, GM-CSF), surgery orradiation. In a preferred embodiment, the AXL MAbs are administered inconjugated form.

AXL ADC formulations are administered via any route capable ofdelivering the antibodies to a tumor cell. Routes of administrationinclude, but are not limited to, intravenous, intraperitoneal,intramuscular, intratumor, intradermal, and the like. Treatmentgenerally involves repeated administration of the AXL ADC preparation,via an acceptable route of administration such as intravenous injection(IV), typically at a dose in the range, including but not limited to,0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 15, 20, or 25 mg/kg body weight. In general, doses in the range of10-1000 mg MAb per week are effective and well tolerated.

Based on clinical experience with the Herceptin® (Trastuzumab) in thetreatment of metastatic breast cancer, an initial loading dose ofapproximately 4 mg/kg patient body weight IV, followed by weekly dosesof about 2 mg/kg IV of the MAb preparation represents an acceptabledosing regimen. Preferably, the initial loading dose is administered asa 90-minute or longer infusion. The periodic maintenance dose isadministered as a 30 minute or longer infusion, provided the initialdose was well tolerated. As appreciated by those of skill in the art,various factors can influence the ideal dose regimen in a particularcase. Such factors include, for example, the binding affinity and halflife of the MAbs used, the degree of AXL expression in the patient, theextent of circulating shed AXL antigen, the desired steady-stateantibody concentration level, frequency of treatment, and the influenceof chemotherapeutic or other agents used in combination with thetreatment method of the invention, as well as the health status of aparticular patient.

Optionally, patients should be evaluated for the levels of AXL in agiven sample (e.g. the levels of circulating AXL antigen and/or AXLexpressing cells) in order to assist in the determination of the mosteffective dosing regimen, etc. Such evaluations are also used formonitoring purposes throughout therapy, and are useful to gaugetherapeutic success in combination with the evaluation of otherparameters (for example, urine cytology and/or ImmunoCyt levels inbladder cancer therapy, or by analogy, serum PSA levels in prostatecancer therapy).

An object of the present invention is to provide AXL ADCs, which inhibitor retard the growth of tumor cells expressing AXL. A further object ofthis invention is to provide methods to inhibit angiogenesis and otherbiological functions and thereby reduce tumor growth in mammals,preferably humans, using such AXL ADCs, and in particular using such AXLADCs combined with other drugs or immunologically active treatments.

VII.) Combination Therapy

In one embodiment, there is synergy when tumors, including human tumors,are treated with AXL ADCs in conjunction with chemotherapeutic agents orradiation or combinations thereof. In other words, the inhibition oftumor growth by a AXL ADC is enhanced more than expected when combinedwith chemotherapeutic agents or radiation or combinations thereof.Synergy may be shown, for example, by greater inhibition of tumor growthwith combined treatment than would be expected from a treatment of onlyAXL ADC or the additive effect of treatment with a AXL ADC and achemotherapeutic agent or radiation. Preferably, synergy is demonstratedby remission of the cancer where remission is not expected fromtreatment either from a AXL ADC or with treatment using an additivecombination of a AXL ADC and a chemotherapeutic agent or radiation.

The method for inhibiting growth of tumor cells using a AXL ADC and acombination of chemotherapy or radiation or both comprises administeringthe AXL ADC before, during, or after commencing chemotherapy orradiation therapy, as well as any combination thereof (i.e. before andduring, before and after, during and after, or before, during, and aftercommencing the chemotherapy and/or radiation therapy). For example, theAXL ADC is typically administered between 1 and 60 days, preferablybetween 3 and 40 days, more preferably between 5 and 12 days beforecommencing radiation therapy and/or chemotherapy. However, depending onthe treatment protocol and the specific patient needs, the method isperformed in a manner that will provide the most efficacious treatmentand ultimately prolong the life of the patient.

The administration of chemotherapeutic agents can be accomplished in avariety of ways including systemically by the parenteral and enteralroutes. In one embodiment, the AXL ADCs and the chemotherapeutic agentare administered as separate molecules. Particular examples ofchemotherapeutic agents or chemotherapy include cisplatin, dacarbazine(DTIC), dactinomycin, mechlorethamine (nitrogen mustard), streptozocin,cyclophosphamide, carmustine (BCNU), lomustine (CCNU), doxorubicin(adriamycin), daunorubicin, procarbazine, mitomycin, cytarabine,etoposide, methotrexate, 5-fluorouracil, vinblastine, vincristine,bleomycin, paclitaxel (taxol), docetaxel (taxotere), aldesleukin,asparaginase, busulfan, carboplatin, cladribine, dacarbazine,floxuridine, fludarabine, hydroxyurea, ifosfamide, interferon alpha,leuprolide, megestrol, melphalan, mercaptopurine, plicamycin, mitotane,pegaspargase, pentostatin, pipobroman, plicamycin, streptozocin,tamoxifen, teniposide, testolactone, thioguanine, thiotepa, uracilmustard, vinorelbine, gemcitabine, chlorambucil, taxol and combinationsthereof.

The source of radiation, used in combination with a AXL ADC, can beeither external or internal to the patient being treated. When thesource is external to the patient, the therapy is known as external beamradiation therapy (EBRT). When the source of radiation is internal tothe patient, the treatment is called brachytherapy (BT).

The above described therapeutic regimens may be further combined withadditional cancer treating agents and/or regimes, for example additionalchemotherapy, cancer vaccines, signal transduction inhibitors, agentsuseful in treating abnormal cell growth or cancer, antibodies (e.g.Anti-CTLA-4 antibodies as described in WO/2005/092380 (Pfizer)) or otherligands that inhibit tumor growth by binding to IGF-1R, and cytokines.

When the mammal is subjected to additional chemotherapy,chemotherapeutic agents described above may be used. Additionally,growth factor inhibitors, biological response modifiers, anti-hormonaltherapy, selective estrogen receptor modulators (SERMs), angiogenesisinhibitors, and anti-androgens may be used. For example, anti-hormones,for example anti-estrogens such as Nolvadex (tamoxifen) or,anti-androgens such as Casodex(4′-cyano-3-(4-fluorophenylsulphonyl)-2-hydroxy-2-methyl-3-′-(trifluoromethyl)propionanilide)may be used.

The above therapeutic approaches can be combined with any one of a widevariety of surgical, chemotherapy or radiation therapy regimens. Thetherapeutic approaches of the invention can enable the use of reduceddosages of chemotherapy (or other therapies) and/or less frequentadministration, an advantage for all patients and particularly for thosethat do not tolerate the toxicity of the chemotherapeutic agent well.

VIII.) Kits/Articles of Manufacture

For use in the laboratory, prognostic, prophylactic, diagnostic andtherapeutic applications described herein, kits are within the scope ofthe invention. Such kits can comprise a carrier, package, or containerthat is compartmentalized to receive one or more containers such asvials, tubes, and the like, each of the container(s) comprising one ofthe separate elements to be used in the method, along with a label orinsert comprising instructions for use, such as a use described herein.For example, the container(s) can comprise an antibody that is or can bedetectably labeled. Kits can comprise a container comprising a DrugUnit.

The kit of the invention will typically comprise the container describedabove and one or more other containers associated therewith thatcomprise materials desirable from a commercial and user standpoint,including buffers, diluents, filters, needles, syringes; carrier,package, container, vial and/or tube labels listing contents and/orinstructions for use, and package inserts with instructions for use.

A label can be present on or with the container to indicate that thecomposition is used for a specific therapy or non-therapeuticapplication, such as a prognostic, prophylactic, diagnostic orlaboratory application, and can also indicate directions for either invivo or in vitro use, such as those described herein. Directions and orother information can also be included on an insert(s) or label(s) whichis included with or on the kit. The label can be on or associated withthe container. A label a can be on a container when letters, numbers orother characters forming the label are molded or etched into thecontainer itself; a label can be associated with a container when it ispresent within a receptacle or carrier that also holds the container,e.g., as a package insert. The label can indicate that the compositionis used for diagnosing, treating, prophylaxing or prognosing acondition, such as a cancer of a tissue set forth in Table I.

The terms “kit” and “article of manufacture” can be used as synonyms.

In another embodiment of the invention, an article(s) of manufacturecontaining compositions, such as antibody(s), or antibody drugconjugates (ADCs) e.g., materials useful for the diagnosis, prognosis,prophylaxis and/or treatment of cancers of tissues such as those setforth in Table I is provided. The article of manufacture typicallycomprises at least one container and at least one label. Suitablecontainers include, for example, bottles, vials, syringes, and testtubes. The containers can be formed from a variety of materials such asglass, metal or plastic. The container can hold amino acid sequence(s),small molecule(s), nucleic acid sequence(s), cell population(s) and/orantibody(s). In another embodiment a container comprises an antibody,binding fragment thereof or specific binding protein for use inevaluating protein expression of AXL in cells and tissues, or forrelevant laboratory, prognostic, diagnostic, prophylactic andtherapeutic purposes; indications and/or directions for such uses can beincluded on or with such container, as can reagents and othercompositions or tools used for these purposes.

The container can alternatively hold a composition that is effective fortreating, diagnosis, prognosing or prophylaxing a condition and can havea sterile access port (for example the container can be an intravenoussolution bag or a vial having a stopper pierceable by a hypodermicinjection needle). The active agents in the composition can be anantibody capable of specifically binding AXL or an antibody drugconjugate specifically binding to AXL.

The article of manufacture can further comprise a second containercomprising a pharmaceutically-acceptable buffer, such asphosphate-buffered saline, Ringer's solution and/or dextrose solution.It can further include other materials desirable from a commercial anduser standpoint, including other buffers, diluents, filters, stirrers,needles, syringes, and/or package inserts with indications and/orinstructions for use.

EXAMPLES

Various aspects of the invention are further described and illustratedby way of the several examples that follow, none of which is intended tolimit the scope of the invention.

Example 1 The AXL Antigen

AXL, otherwise know as Tyrol, UFO, and ARK (as well as, GenBankAccession No.: NM_021913) is a protein that encodes a receptor tyrosinekinase with high amino acid similarity to the human trk, eph, eck, andros proteins, and insulinlike growth factor 1 receptor. The proteinencoded by this gene belongs to a transmembrane receptor tyrosine kinase(TRKs) family which includes the Rse/Tyro3 and MER receptors. Thisfamily is characterized by a unique extracellular composition ofimmunoglobulin-like fibronectin III-like domains. These structuralfindings suggest Axl family members may be involved in both celladhesion and intracellular signalling. See, Sainaghi, et. al., J. Cell.Phys. 204:36-44 (2005). See also, O'Bryan, et. al., J. Bio. Chem., Vol.270 no. 2, pp. 551-557 (1995). Also, it has been noted that Axl isexpressed in multiple cancers such as lung, breast, ovarian, thyroid,gastric, kidney, colon, and myeloid leukemias. For further reference,see Shieh, et. al., Neoplasia, vol 7, No. 12 pp 1058-1064 (December2005); Berclaz, et. al. Annals of Oncology, vol. 12 pp. 819-824 (2001);Sun, et. al. Oncology 2004; 66:450-457 (2004); Ito, et. al., Thyroid,vol. 9:No. 6, pp. 563-567 (1999); Wu, et. al., AntiCancer Res., vol. 22,pp. 1071-1078 (2002); Chung, et. al. DNA and Cell Bio., Vol. 22:No. 8,pp. 533-540 (2003); Craven, et.al., Int. J. Cancer, vol. 60 pp. 791-797(1995). The AXL cDNA is 4,743 bp in length and encodes a 894 amino acidORF (See, FIG. 1). For exemplary embodiments of the AXL antigen, seeFIG. 1.

Example 2 Generation of AXL Monoclonal Antibodies (MAbs)

In one embodiment, diagnostic Monoclonal Antibodies (“mAbs”) to AXLcomprise those that react with epitopes specific for AXL that would bindto AXL expressed on cells in frozen or paraffin embedded, formalinfixed, tissue sections prepared from patient biopsies. Immunogens forgeneration of such MAbs include those designed to encode or contain theAXL protein sequence. Immunogens include peptides, recombinant proteins,cells that endogenously express AXL or those that have been engineeredto express AXL (such as 293T-AXL).

MAbs to AXL were generated using either VelocImmune® mice or balb/cmice. The mAb designated V77-2a37.1 was generated after immunizingvelocimmune mice with recombinant 293T cells expressing AXL. The AXLMAb, V77-2a37.1 specifically binds to AXL expressing cells (recombinantand endogenous) and AXL expressing tumor cells in frozen or paraffinembedded, formalin fixed, tissue sections prepared from patientbiopsies.

DNA coding sequences for AXL MAb V77-2a37.1 was determined afterisolating mRNA from the respective hybridoma cells with Trizol reagent(Life Technologies, Gibco BRL).

Anti-AXL V77-2a37.1 heavy and light chain variable nucleic acidsequences were sequenced from the hybridoma cells using the followingprotocol. V77-2a37.1 secreting hybridoma cells were lysed with Trizolreagent (Life Technologies, Gibco BRL). Total RNA was purified andquantified. First strand cDNAs was generated from total RNA with oligo(dT)12-18 priming using the Gibco-BRL Superscript Preamplificationsystem. First strand cDNA was amplified using human immunoglobulinvariable heavy chain primers, and human immunoglobulin variable lightchain primers. PCR products were sequenced and the variable heavy andlight chain regions determined.

The nucleic acid and amino acid sequences of the full length heavy andlight chains are listed in FIGS. 2A and 2B and FIGS. 3A and 3B.Alignment of V77-2a37.1 MAbs to human Ig germline is set forth in FIG.4A-4B.

Example 3 Human Clinical Trials for the Treatment and Diagnosis of HumanCarcinomas through Use of AXL ADCs

AXL ADCs are used in accordance with the present invention whichspecifically bind to AXL, and are used in the treatment of certaintumors, preferably those listed in Table I. In connection with each ofthese indications, two clinical approaches are successfully pursued.

I.) Adjunctive therapy: In adjunctive therapy, patients are treated withAXL ADCs in combination with a chemotherapeutic or anti-neoplastic agentand/or radiation therapy or a combination thereof. Primary cancertargets, such as those listed in Table I, are treated under standardprotocols by the addition of AXL ADCs to standard first and second linetherapy. Protocol designs address effectiveness as assessed by thefollowing examples, including but not limited to, reduction in tumormass of primary or metastatic lesions, increased progression freesurvival, overall survival, improvement of patients health, diseasestabilization, as well as the ability to reduce usual doses of standardchemotherapy and other biologic agents. These dosage reductions allowadditional and/or prolonged therapy by reducing dose-related toxicity ofthe chemotherapeutic or biologic agent. AXL ADCs are utilized in severaladjunctive clinical trials in combination with the chemotherapeutic oranti-neoplastic agents.

II.) Monotherapy: In connection with the use of the AXL ADCs inmonotherapy of tumors, the AXL ADCs are administered to patients withouta chemotherapeutic or anti-neoplastic agent. In one embodiment,monotherapy is conducted clinically in end-stage cancer patients withextensive metastatic disease. Protocol designs address effectiveness asassessed by the following examples, including but not limited to,reduction in tumor mass of primary or metastatic lesions, increasedprogression free survival, overall survival, improvement of patientshealth, disease stabilization, as well as the ability to reduce usualdoses of standard chemotherapy and other biologic agents.

Dosage

Dosage regimens may be adjusted to provide the optimum desired response.For example, a single bolus may be administered, several divided dosesmay be administered over time or the dose may be proportionally reducedor increased as indicated by the exigencies of the therapeuticsituation. It is especially advantageous to formulate parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the mammaliansubjects to be treated; each unit containing a predetermined quantity ofactive compound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on (a) the unique characteristics of the antibody and/or ADCand the particular therapeutic or prophylactic effect to be achieved,and (b) the limitations inherent in the art of compounding such anactive compound for the treatment of sensitivity in individuals.

An exemplary, non limiting range for a therapeutically effective amountof an AXL ADC administered in combination according to the invention isabout 0.5 to about 10 mg/kg, about 1 to about 5 mg/kg, at least 1 mg/kg,at least 2 mg/kg, at least 3 mg/kg, or at least 4 mg/kg. Other exemplarynon-limiting ranges are for example about 0.5 to about 5 mg/kg, or forexample about 0.8 to about 5 mg/kg, or for example about 1 to about 7.5mg/kg. The high dose embodiment of the invention relates to a dosage ofmore than 10 mg/kg. It is to be noted that dosage values may vary withthe type and severity of the condition to be alleviated, and may includesingle or multiple doses. It is to be further understood that for anyparticular subject, specific dosage regimens should be adjusted overtime according to the individual need and the professional judgment ofthe person administering or supervising the administration of thecompositions, and that dosage ranges set forth herein are exemplary onlyand are not intended to limit the scope or practice of the claimedcomposition.

Clinical Development Plan (CDP)

The CDP follows and develops treatments of AXL ADCs in connection withadjunctive therapy or monotherapy. Trials initially demonstrate safetyand thereafter confirm efficacy in repeat doses. Trials are open labelcomparing standard chemotherapy with standard therapy plus AXL ADCs. Aswill be appreciated, one non-limiting criteria that can be utilized inconnection with enrollment of patients is AXL expression levels in theirtumors as determined by biopsy.

As with any protein or antibody infusion-based therapeutic, safetyconcerns are related primarily to (i) cytokine release syndrome, i.e.,hypotension, fever, shaking, chills; (ii) the development of animmunogenic response to the material (i.e., development of humanantibodies by the patient to the antibody therapeutic, or HAMAresponse); and, (iii) toxicity to normal cells that express AXL.Standard tests and follow-up are utilized to monitor each of thesesafety concerns. AXL ADCs are found to be safe upon humanadministration.

Example 4 Detection of AXL Protein in Cancer Patient Specimens by IHC

Expression of AXL protein by immunohistochemistry was tested in tumorspecimens from sarcoma, pancreatic, melanoma, ovarian, and lung cancersamples. Briefly, formalin fixed, paraffin wax-embedded tissues were cutinto four (4) micron sections and mounted on glass slides. The sectionswere de-waxed, rehydrated, and treated with Novocastra Bond EpitopeRetrieval Solution 2 (Leica Biosystems, Buffalo Grove, Ill.) in theBOND-MAX automated IHC staining system (Leica Biosystems, Buffalo Grove,Ill.) for thirty (30) minutes at 100° C. then placed for twelve (12)minutes at room temperature. Sections were then incubated with eithermonoclonal mouse anti-AXL antibody denoted V77-2a37.1, or an isotypecontrol. Subsequently, the sections were treated with the NovocastraBond Polymer Refine Detection System which consists of incubation in apost-primary rabbit anti-mouse IgG reagent followed by incubation with apolymer anti-rabbit poly-HRP-IgG reagent (Leica Biosystems, BuffaloGrove, Ill.). Sections were then treated with 3% hydrogen peroxidesolution to inactivate endogenous peroxidase activity. Chromogensubstrate visualization was developed using the DAB refine kit (LeicaBiosystems, Buffalo Grove, Ill.), nuclei were stained using hematoxylin,and slides were scanned and analyzed on the Aperio ePathology Scanscopeimaging system (Leica Biosystems, Vista, Calif.). Specific staining wasdetected in patient specimens using the AXL-specific V77-2a37.1antibody, as indicated by the brown staining. (See, FIGS. 5A, C, E, G,and I). In contrast, the isotype control antibody did not stain thetumor specimen. (See, FIGS. 5B, D, F, H, and J).

The results show expression of AXL in the tumor cells of patient sarcoma(FIG. 5A), pancreatic (FIG. 5C)), melanoma (FIG. 5E), ovarian (FIG. 5G),and lung cancer (FIG. 5I) tissues. These results indicate that AXL isexpressed in human cancers and that antibodies directed to this antigenare useful for diagnostic and prognostic purposes. (FIG. 5A-FIG. 5J).

Example 5 Epitope Mapping of V77-2a37.1 MAb

It is known in the art if the epitopes of two antibodies areoverlapping, or are in close proximity to one another, then it isanticipated that the antibodies would block each other to an extentdetermined by the proximity of their epitopes. An antibody competitionexperiment, in which one antibody is labeled and challenged with a molarexcess of unlabeled antibody, then reacted with immobilized targetprotein, is a facile method for determining whether two antibodies bindto epitopes located in close proximity to one another.

An antibody competition experiment was undertaken to determine whetherthe anti-AXL antibody of the invention (i.e. V77-2a37.1) interfere withanother MAbs which bind AXL, and thus bind to overlapping epitopes.

In this experiment, test antibodies were first biotinylated. Thebiotinylated antibodies were then challenged with a panel of unlabeledantibodies in an AXL binding assay, all present at a 50 fold molarexcess to the biotinylated antibodies. The unlabeled antibody panelincluded the unlabeled versions of the two test antibodies, a thirdantibody directed to AXL from Cell Signaling, and an appropriate isotypecontrol. After a two hour incubation period, in which the mixture ofbiotinylated and unlabeled antibodies were reacted with recombinanthuman AXL, immobilized onto an ELISA plate, unbound antibody was removedby washing, and immobilized biotin was detected with Streptavidin-HRPfollowed by TMB substrate solution.

It was expected that the unlabeled version of v77-2a37.1 would block thebiotinylated version of itself. The same result was anticipated for theunlabeled and biotinylated versions of other AXL MAbs. However, if theunlabeled version of v77-2a37.1 was found to block the biotinylatedversion of the other AXL MAbs, or vice versa, the conclusion would bethat these antibodies compete with each other, due to their epitopes onthe AXL protein being in close proximity. The results obtained with theCell Signaling antibody would indicate if the epitope of this antibodywas distant or in close proximity to that of v77-2a37.1.

Materials and Methods

The protein and antibodies used in this experiment are summarized inTable IV.

BIOTINYLATION OF ANTIBODIES: Aliquots of AXL antibodies v77-2a37.1 andM77-297b81.1.1 were biotinylated using a 20-fold molar ratio ofSulfo-NHS-LC-Biotin (Thermo-Fisher, Waltham, Mass.) for 2 hours at roomtemperature. Unincorporated biotin was removed by exhaustive dialysisagainst PBS.

IMMOBILIZATION OF RECOMBINANT AXL: Recombinant human Axl-tag 5 proteinwas first diluted to 0.5 μg/mL in carbonate buffer. Aliquots of thediluted protein solution (50 μL) were pipetted into the wells of a 96well Nunc Maxisorp ELISA plate (Thermo-Fisher, Waltham, Mass.). Theplate was covered and incubated overnight at room temperature toimmobilize the protein. The plate was then aspirated and washed 3 timeswith PBST (PBS plus 0.05% Tween-20), 200 μL per well per wash cycle. Theplate was blocked with 200 μL blocking solution (PBS plus 3% non-fatdried milk) for 1 hour at room temperature.

ANTIBODY COMPETITION: The unlabeled antibodies were diluted to aconcentration of 100 μg/mL in PBST plus 3% non-fat dried milk, while thetwo biotinylated antibodies were diluted to a concentration of 2 μg/mLin PBST plus 3% non-fat dried milk. Aliquots (25 μL) of each of theunlabeled antibodies were pipetted into designated wells of the coatedplate, followed by 25 μL aliquots of the biotinylated antibodies. Thesolutions were mixed well, and incubated in the plate for 2 hours atroom temperature. The plate was washed 3 times with PBST. Boundbiotinylated antibody was detected with 50 μL Streptavidin-HRP (SouthernBiotech, Birmingham, Ala.); diluted 1 to 5000 in PBST with 3% non-fatdried milk; 1 hour at room temperature). The plate was then washed 3times, and bound HRP was detected with 70 μL TMB (20 minutes at roomtemperature), followed by 50 μL stop solution. The optical density ofthe plate was then measured at 650 nm.

RESULTS: The results show that in the absence of any unlabeled antibody,the biotinylated versions of both V77-2a37.1 and M77-297b81.1.1 MAbsproduced a robust signal with the AXL-coated ELISA plate. This resultindicated that the plate was well coated and that the biotinylationreaction produced good results.

Furthermore, the isotype control antibody included in this experiment,cmlys-1c3.1, did not interfere with the ability of the two biotinylatedantibodies to bind to AXL, indicating that the isotype control does notcompete with either of the two biotinylated antibodies.

As anticipated, the unlabeled version of antibody V77-2a37.1, present ata 50 fold molar excess of the biotinylated version, competes with thebiotinylated version of itself for binding to immobilized AXL. Likewise,the unlabeled version of M77-297b81.1.1 competes with the biotinylatedversion of itself. However, when the biotinylated version of V77-2a37.1is presented with a 50 fold molar excess of M77-297b81.1.1, nocompetition is observed, indicating that the epitopes of these twoantibodies are indeed distinct. In support of this observation, when thebiotinylated version of M77-297b81.1.1 is presented with a 50 fold molarexcess of V77-2a37.1, no competition is observed (Table V and Table VI).

Additionally, this experiment also examined the ability of the CellSignaling rabbit monoclonal anti-AXL antibody to compete withbiotinylated V77-2a37.1 and biotinylated M77-297b81.1.1 MAb. The resultspresented show indicate that the Cell Signaling rabbit monoclonalanti-AXL antibody has a small impact (3.3% competition) on the abilityof biotinylated V77-2a37.1 to bind to AXL, and a similar small impact(1.5%) on the ability of biotinylated M77-297b81.1.1 to bind to AXL.These results suggest the potential of a di minimus degree of overlapbetween the epitopes of Cell Signaling rabbit monoclonal anti-AXLantibody and the two test antibodies (Table VI).

Example 6 Evaluation of C89E7 MAb and AF154 MAb as anImmunohistochemistry Reagent for AXL Detection Using Cell Block Samples

In this experiment, three (3) cell block samples, Human lung cancer celllines NCI-H292 and HCC827 (purchased from American Type CultureCollection), and NCI-H727 (purchased from European Collection of CellCulture) were fixed in 10% phosphate buffered formalin overnight aftercells were cultured in RPMI1640 medium (Thermo Fisher Scientific,Waltham, Mass.), supplemented with 10% Fetal Bovine Serum(Sigma-Aldrich, St. Louis, Mo.), and centrifuged and washed. Then thecell pellets were mixed with O.C.T. Compound (Sakura Finetek Japan) toobtain cell blocks which were subsequently embedded in paraffin.

After cutting the cell blocks and mounting on glass slides, AXLexpression in the sections was confirmed by RNA in situ hybridization(RNA ISH) using RNA ISH kit(QuantiGene ViewRNA ISH tissue 1-Plex Assaykit (Affymetrix, Santa Clara, Calif.) and QuantiGene ViewRNA ChromogenicSignal Amp kit (Affymetrix, Santa Clara, Calif.) with QuantiGene ViewRNATYPE1 Probe Set Human AXL (Affymetrix, Santa Clara, Calif.) according tothe provided user manual). To visualize the AXL mRNAs labelled with thealkaline phosphatase (AP), WarpRed Chromogen Kit (Biocare Medical,Concord, Calif.) was used as the chromogen according to the protocolincluded in the kit. Then the slides were counterstained withhematoxylin, air-dried, cleared in xylene, and mounted with permanentmounting medium Entellan new reagent (Merck, Darmstadt, Germany).Specific signal of AXL expression was observed in the NCI-H292 cells(FIG. 6A(i)) whereas virtually no signal were observed in HCC827 (FIG.6A(ii)) and NCI-H727 cells (FIG. 6A(iii), indicating NCI-H292 cell linewas AXL positive, but both HCC827 and NCI-H727 cell lines were AXLnegative.

Next, two anti-AXL antibodies, the Cell Signaling rabbit monoclonalantibody [C89E7] (Cell signaling Technologies, Danvers, Mass.) and theR&D goat polyclonal antibody [AF154] (R&D systems, Minneapolis, Minn.)were evaluated using the cell block samples by immunohistochemistry(IHC). Briefly, the sections of the cell blocks were de-waxed andrehydrated, and antigen retrieval was performed by autoclaving (121° C.for 10 minutes) or microwaving (100° C. for 15 minutes), and thenendogenous peroxidase was inactivated with 3% H₂O₂-methanol. The slideswere then blocked with TBS containing 1% of BSA and incubated with 1:100diluted antibodies in the same buffer. After three (3) times of washwith TBST (TBS plus 0.05% Tween 20), the AXL in the slides was detectedand visualized with the second antibody and 3,3′-Diaminobenzidine,tetrahydrochloride (DAB) reagent listed in Table VIII, before beingcounterstained with Mayer's hematoxylin. The detailed conditions used inthe IHC experiments are described in Table VIII. The results of IHCstaining of AXL on these three cell blocks were shown in FIG.6B(i)-(vi). The IHC for AXL with anti-AXL monoclonal antibody [C89E7] inNCI-H292 cells showed intense stainings (FIG. 6B(i)), but virtually nostainings were observed in HCC827 (FIG. 6B(ii)) and NCI-H727 cells (FIG.6B(iii)), which was consistent with the results of AXL expressionobservation obtained by RNA ISH experiments, whereas the marginal orweak IHC staining signal were observed in HCC827 (FIG. 6B(v)) andNCI-H727 (FIG. 6B(vi)) cells by anti-AXL polyclonal antibody [AF154],which was not consistent with the RNA ISH results. These observationssuggested that anti-AXL monoclonal antibody [C89E7] appeared to be moresuitable as a IHC reagent than anti-AXL polyclonal antibody [AF154]since anti-AXL monoclonal antibody [C89E7] did not show non-specificsignal as observed in these experiments using these three cell blocks.

Example 7 Comparative Immunohistochemistry in Selected AXL RNA (+) (−)FFPE Samples Using anti-AXL V77-2a37.1 MAb, M77-297b81.1.1 MAb, andC89E7 MAb

Non-specific IHC staining with anti-AXL antibodies V77-2a37.1,M77-297b81.1.1 (ATCC Designation PTA-122092), and AXL (C89E7) wasevaluated in AXL-negative tumor specimens (measured by qPCR) from breast(FIGS. 7A-7D), hepatocellular (FIGS. 7E-7H), and colon (FIGS. 7I-7L)carcinoma samples.

Briefly, formalin fixed, paraffin wax-embedded tissues were cut into 4micron sections and mounted on glass slides. The sections were de-waxed,rehydrated and treated with Novocastra Bond Epitope Retrieval Solution 2(Leica Biosystems, Buffalo Grove, Ill.) in the BOND-MAX automated IHCstaining system (Leica Biosystems, Buffalo Grove, Ill.) for 30 minutesat 100° C. then for 12 minutes at room temperature. Sections were thenincubated with monoclonal anti-AXL antibody V77-2a37.1, M77-297b81.1,monoclonal rabbit anti-AXL antibody Axl (C89E7) (Cell SignalingTechnology, Danvers, Mass.), or an isotype control. Subsequently, thesections were treated with the Novocastra Bond Polymer Refine DetectionSystem which consists of incubation in a post-primary rabbit anti-mouseIgG reagent followed by incubation with a polymer anti-rabbitpoly-HRP-IgG reagent (Leica Biosystems, Buffalo Grove, Ill.). Sectionswere then treated with 3% hydrogen peroxide solution to inactivateendogenous peroxidase activity. Chromogen substrate visualization wasdeveloped using the DAB refine kit (Leica Biosystems, Buffalo Grove,Ill.), nuclei were stained using hematoxylin, and slides were scannedand analyzed on the Aperio ePathology Scanscope imaging system (LeicaBiosystems, Vista, Calif.).

The results show the limited or absence of non-specific staining wasobserved in all of the tumor specimens using the AXL-specific V77-2a37.1(FIGS. 7A, 7E, 7I) and M77-297b81.1.1 (FIGS. 7B, 7F, 7J) antibodies, asindicated by the lack of brown staining. Furthermore, the isotypecontrol antibody also did not exhibit non-specific staining in the tumorspecimens (FIGS. 7D, 7H, 7L). In contrast, the commercially availableanti-AXL antibody, AXL (C89E7), exhibited noticeable brown staining(FIGS. 7C, 7G, 7K, see arrows) in the tumor specimens with limited AXLmRNA expression.

The results in FIGS. 7A-7L indicate that V77-2a37.1 and M77-297b81.1.1are superior IHC antibodies due to the absence of non-specific stainingin AXL mRNA negative tissue samples.

Throughout this application, various website data content, publications,patent applications and patents are referenced. (Websites are referencedby their Uniform Resource Locator, or URL, addresses on the World WideWeb.) The disclosures of each of these references are herebyincorporated by reference herein in their entireties.

The present invention is not to be limited in scope by the embodimentsdisclosed herein, which are intended as single illustrations ofindividual aspects of the invention, and any that are functionallyequivalent are within the scope of the invention. Various modificationsto the models and methods of the invention, in addition to thosedescribed herein, will become apparent to those skilled in the art fromthe foregoing description and teachings, and are similarly intended tofall within the scope of the invention. Such modifications or otherembodiments can be practiced without departing from the true scope andspirit of the invention.

Tables

TABLE I Tissues/Cells that express AXL when malignant. Lung; Ovarian;Melanoma; Pancreatic; Sarcoma.

TABLE II Amino Acid Abbreviations SINGLE LETTER THREE LETTER FULL NAME FPhe phenylalanine L Leu leucine S Ser serine Y Tyr tyrosine C Cyscysteine W Trp tryptophan P Pro proline H His histidine Q Gln glutamineR Arg arginine I Ile isoleucine M Met methionine T Thr threonine N Asnasparagine K Lys lysine V Val valine A Ala alanine D Asp aspartic acid EGlu glutamic acid G Gly glycine

TABLE III Amino Acid Substitution Matrix Adapted from the GCG Software9.0 BLOSUM62 amino acid substitution matrix (block substitution matrix).The higher the value, the more likely a substitution is found inrelated, natural proteins. A C D E F G H I K L M N P Q R S T V W Y . 4 02 1 2 0 2 1 1 1 1 2 1 1 1 1 0 0 −3 −2 A 9 3 4 2 3 3 1 3 1 1 3 3 3 3 1 11 2 2 C 6 2 3 1 1 3 1 4 3 1 1 0 2 0 1 3 4 3 D 5 −3 −2 0 −3 1 −3 −2 0 −12 0 0 −1 −2 −3 −2 E 6 −3 −1 0 −3 0 0 3 4 3 3 2 2 1 1 3 F 6 2 4 2 4 3 0 22 2 0 2 3 2 3 G 8 −3 −1 −3 −2 1 −2 0 0 −1 −2 −3 −2 2 H 4 −3 2 1 3 3 3 32 1 3 3 1 I 5 −2 −1 0 −1 1 2 0 −1 −2 −3 −2 K 4 2 3 3 2 2 2 1 1 2 1 L 5−2 −2 0 −1 −1 −1 1 −1 −1 M 6 −2 0 0 1 0 −3 −4 −2 N 7 1 2 1 1 2 4 3 P 5 10 −1 −2 −2 −1 Q 5 1 1 3 3 2 R 4 1 −2 −3 −2 S 5 0 −2 −2 T 4 −3 −1 V 11 2W 7

TABLE IV Description of the Recombinant AXL Protein and Antibodies usedin the Competition Experiment (Example 5). Stock ConcentrationDescription Name Source Lot (mg/ml) Recombinant Human Axl ProteinAxl-tag 5 Agensys, Inc. DT1040 1.67 Mouse anti-Hu Axl monoclonalv77-2a37.1 Agensys, Inc. RC2933 2.05 antibody (purified) Mouse anti-HuAxl monoclonal M77-297b81.1.1 Agensys, Inc. RC5481 5.66 antibody(purified) Rabbit anti-Hu Axl monoclonal AXL(C89E7) Cell Signaling Lot 40.16 antibody (purified) Mouse anti-Lysozyme monoclonal cmlys-1c3.1Agensys, Inc. RC3265 2.16 antibody (purified)

TABLE V Summary of the Antibody Competition Experiment (Example 5).Biot-v77-2a37.1 Biot-M77-297b81.1.1 Unlabeled (1 μg/mL) (1 μg/mL)Antibody OD % OD % (50 μg/mL) (650 nm) competition (650 nm) competitionv77-2a37.1 0.04 97.7% 2.15 0.6% M77-297b81.1.1 1.88 0.0% 0.04 98.2%AXL(C89E7) 1.81 3.3% 2.13 1.5% cmlys-1c3.1 1.88 0.0% 2.31 0.0% biot-Abonly 1.87 2.16 blank 0.04 0.04

TABLE VII Positions CDR-L1, CDR-L2, CDR-L3 and CDR-H1,CDR-H2, CDR-H3 as identified by the Kabat,Chothia, and Contact schemes, respectively.For CDR-H1, residue numbering is given listedusing both the Kabat and Chothia numbering schemes. CDR Kabat ChothiaContact CDR-L1 L24-L34 L24-L34 L30-L36 RASQSISNWLA RASQSISNWLA SNWLAWY(SEQ ID NO: 11) (SEQ ID NO: 14) (SEQ ID NO: 17) CDR-L2 L50-L56 L50-L56L46-L55 KASSLES KASSLES LLIYKASSLE (SEQ ID NO: 12) (SEQ ID NO: 15)(SEQ ID NO: 18) CDR-L3 L89-L97 L89-L97 L89-L96 QQYNSYYT QQYNSYYT QQYNSYY(SEQ ID NO: 13) (SEQ ID NO: 16) (SEQ ID NO: 19) CDR-H1* H31-H37 H26-H34H30-H35B NSSYHWG GVSITNSSY TNSSYHWG (SEQ ID NO: 20) (SEQ ID NO: 24)(SEQ ID NO: 28) CDR-H1** H31-H35 H26-H32 H30-H35 NSSYHWG GVSITNSSYTNSSYHWG (SEQ ID NO: 21) (SEQ ID NO: 25) (SEQ ID NO: 29) CDR-H2 H50-H65H52-H56 H47-H58 SIFYNGNTFFNPSLKS FYNGN WIGSIFYNGNTF (SEQ ID NO: 22)(SEQ ID NO: 26) (SEQ ID NO: 30) CDR-H3 H95-H102 H95-H102 H93-H101QDNWNFRHYFNY QDNWNFRHYFNY ERQDNWNFRHYFN (SEQ ID NO: 23) (SEQ ID NO: 27)(SEQ ID NO: 31) *Kabat Numbering **Chothia Numbering

TABLE VIII IHC reagents and protocols Antibodies Antigen retrievalCondition Detection System/Condition Chromogen/Condition AXL [C89E7]Citrate buffer, 121° C., 1:100 EnVision+ System-HRP RT, ImmPACT ™ 2-fold(Cell Signaling pH 6.0 10 min RT Labelled Polymer Anti- 30 min DAB(Vector dilution Technology) 2 hr. Rabbit (DAKO) Laboratories) 2 min AXL[AF1541] Immunosaver 100° C., 1:100 anti-Goat IgG H&L 1:200, DAB TRIStablet 1 min (R&D Systems) (Nisshin EM) 15 min RT (Rabbit) Antibody, RT(MUTO PURE 2 hr. Peroxidase conjugated 30 min CHEMICALS (Rockland CO.,LTD. Immunochemicals Inc.)

1. An antibody or antigen binding fragment thereof which binds to AXL protein, wherein the antibody or fragment comprises a heavy chain variable region comprising complementarity determining regions (CDRs) consisting of the amino acid sequences of the CDRs in the heavy chain variable region sequence set forth in SEQ ID NO: 8 and a light chain variable region comprising complementarity determining regions (CDRs) consisting of the amino acid sequences of the CDRs in the light chain variable region sequence set forth in SEQ ID NO:
 10. 2. An antibody or antigen binding fragment of claim 1, wherein the antibody or antigen binding fragment comprises a heavy chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 8, and a light chain variable region consisting of the amino acid sequence set forth in SEQ ID NO:
 10. 3. An antibody of claim 1, wherein the antibody comprises a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 7, and a light chain consisting of the amino acid sequence set forth in SEQ ID NO:
 9. 4. The antigen binding fragment of claim 1, wherein the fragment is an Fab, F(ab′)₂, Fv or scFv fragment.
 5. The antibody or antigen binding fragment of claim 1, wherein the antibody or antigen binding fragment is a fully human antibody.
 6. The antibody or antigen binding fragment of claim 1, which the antibody or antigen binding fragment is recombinantly produced.
 7. The antibody or antigen binding fragment of claim 1, wherein the antibody or antigen binding fragment is conjugated to an imaging agent.
 8. The antibody or antigen binding fragment of claim 1, wherein the antibody or antigen binding fragment is used as a companion diagnostic (CDx) to screen patients for the treatment and management of cancer.
 9. A method of detecting AXL expression in a subject comprising a step of contacting the antibody or antigen binding fragment of claim 1 with a sample obtained from the subject.
 10. The method of claim 9, which comprises a step of obtaining a sample from the subject.
 11. The method of claim 9, wherein the subject is a cancer patient.
 12. The method of claim 11, wherein wherein the cancer is selected from the group consisting of sarcoma, pancreatic cancer, melanoma, ovarian cancer, and lung cancer.
 13. A method for identifying a cancer patient which is positive for AXL expression, which comprises a step of contacting the antibody or antigen binding fragment of claim 1 with a sample obtained from the subject.
 14. The method of claim 13, which comprises a step of obtaining a sample from the subject.
 15. The method of claim 13, wherein the cancer is selected from the group consisting of sarcoma, pancreatic cancer, melanoma, ovarian cancer, and lung cancer.
 16. The method of claim 13, wherein the cancer patient has undergone treatment by EGFR inhibitor.
 17. The method of claim 13, wherein the cancer is resistant to EGFR inhibitor.
 18. A kit for detecting AXL expression comprising the antibody or antigen binding fragment of claim
 1. 